UspA2 protein constructs and uses thereof

ABSTRACT

The present invention relates to compositions comprising Moraxella catarrhalis (M. catarrhalis) Ubiquitous surface protein A2 (UspA2). More particularly, the present application relates to UspA2 protein constructs and immunogenic compositions comprising the constructs, vaccines comprising such immunogenic compositions and therapeutic uses of the same. The invention further relates to compositions comprising UspA2 in combination with at least one antigen from Haemophilus influenzae, immunogenic compositions comprising the antigens, vaccines comprising such immunogenic compositions and therapeutic uses of the same.

FIELD OF THE INVENTION

The present invention relates to compositions comprising Moraxellacatarrhalis (M. catarrhalis, M. cat.) Ubiquitous surface protein A2(UspA2). More particularly, the present application relates to UspA2protein constructs and immunogenic compositions comprising theconstructs, vaccines comprising such immunogenic compositions andtherapeutic uses of the same.

BACKGROUND OF THE INVENTION

Ubiquitous surface protein A2 (UspA2) is a trimeric autotransporter thatappears as a lollipop-shared structure in electron micrographs (Hoiczyket al. EMBO J. 19: 5989-5999 (2000)). It is composed of a N-terminalhead, followed by a stalk which ends by an amphipathic helix and aC-terminal membrane domain. (Hoiczyk et al. EMBO J. 19: 5989-5999(2000)). UspA2 contains a very well conserved domain (Aebi et al.,Infection & Immunity 65(11) 4367-4377 (1997)), which is recognized by amonoclonal antibody that was shown protective upon passive transfer in amouse Moraxella catarrhalis challenge model (Helminnen et al. J InfectDis. 170(4): 867-72 (1994)).

UspA2 has been shown to interact with host structures and extracellularmatrix proteins like fibronectin (Tan et al., J Infect Dis. 192(6):1029-38 (2005)) and laminin (Tan et al., J Infect Dis. 194(4): 493-7(2006)), suggesting it can play a role at an early stage of Moraxellacatarrhalis infection.

UspA2 also seems to be involved in the ability of Moraxella catarrhalisto resist the bactericidal activity of normal human serum. (Attia A S etal. Infect Immun 73(4): 2400-2410 (2005)). It (i) binds the complementinhibitor C4bp, enabling Moraxella catarrhalis to inhibit the classicalcomplement system, (ii) prevents activation of the alternativecomplement pathway by absorbing C3 from serum and (iii) interferes withthe terminal stages of the complement system, the Membrane AttackComplex (MAC), by binding the complement regulator protein vitronectin.(de Vries et al., Microbiol Mol Biol Rev. 73(3): 389-406 (2009)).

Moraxella catarrhalis is an important and common respiratory pathogenthat has been associated with increased risk of exacerbations in chronicobstructive pulmonary disease (COPD) in adults. (Sateesh et al., Journalof Chronic Obstructive Pulmonary Disease 3:109-115 (2006)).

A need for vaccines for Moraxella catarrhalis exists.

BRIEF SUMMARY OF THE INVENTION

As a first aspect, the present invention provides the proteins offormula (I).A-(R₁)_(m)—(B)_(n)  (formula I)

wherein:

A is UspA2 from Moraxella catarrhalis or an immunogenic fragmentthereof;

R₁ is an amino acid;

m is 0, 1 or 2;

B is histidine; and

n is 0, 1, 2, 3, 4, 5 or 6.

As a second aspect, the present invention provides immunogeniccompositions comprising proteins of formula (I) and proteins of theinvention. The composition may further comprise a pharmaceuticallyacceptable adjuvant. The composition may comprise an excipient.

In a third aspect, the present invention provides a method for thetreatment or prevention of a condition or disease caused wholly or inpart by Moraxella catarrhalis. The method comprises administering to asubject in need thereof a therapeutically effective amount of a proteinof formula (I) or a protein of the invention.

In a fourth aspect, the present invention provides a method for thetreatment or prevention of otitis media. The method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of a protein of formula (I) or a protein of the invention.

In a fifth aspect, the present invention provides a method for thetreatment or prevention of exacerbations in chronic obstructivepulmonary disease. The method comprises administering to a subject inneed thereof a therapeutically effective amount of a protein of formula(I) or a protein of the invention.

In a sixth aspect, the present invention provides a method for thetreatment or prevention of pneumonia. The method comprises administeringto a subject in need thereof a therapeutically effective amount of aprotein of formula (I) or a protein of the invention.

In a seventh aspect, the present invention provides a pharmaceuticalcomposition comprising a protein of formula (I) or a protein of theinvention for use in the treatment or prevention of a condition ordisease caused wholly or in part by Moraxella catarrhalis.Pharmaceutical compositions may further comprise a pharmaceuticallyacceptable adjuvant.

In an eighth aspect, the present invention provides nucleic acidsencoding the proteins of the invention.

In a ninth aspect, the present invention provides a process of producingnucleic acids of the invention.

In a tenth aspect, the present invention provides a compositioncomprising at least one antigen from Moraxella catarrhalis and at leastone antigen from Haemophilus influenzae. The composition may furthercomprise a pharmaceutically acceptable adjuvant. The composition maycomprise an excipient.

In an additional aspect, the present invention provides a method for thetreatment or prevention of a condition or disease caused wholly or inpart by Moraxella catarrhalis and/or Haemophilus influenzae. The methodcomprises administering to a subject in need thereof a therapeuticallyeffective amount of a protein of formula (I) or a protein of theinvention.

In a further aspect, the present invention provides a method for thetreatment or prevention of exacerbations in chronic obstructivepulmonary disease. The method comprises administering to a subject inneed thereof a therapeutically effective amount of a protein of formula(I) or a protein of the invention and a therapeutically effective amountof at least one antigen from Haemophilus influenzae.

The present invention also provides a pharmaceutical compositioncomprising a protein of formula (I) or a protein of the invention foruse in the treatment or prevention of a condition or disease causedwholly or in part by Moraxella catarrhalis in combination with at leastone antigen from Haemophilus influenzae. Pharmaceutical compositions mayfurther comprise a pharmaceutically acceptable adjuvant.

Further aspects of the present invention are described in the detaileddescription of particular embodiments, examples and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A typical fermentation profile with the High Cell DensityInduction (HCDI) processes and the parameters monitored during 20L-scale fed-batch fermentation.

FIG. 2: A typical fermentation profile with the Low Cell DensityInduction (LCDI) processes and the parameters monitored during 20L-scale fed-batch fermentation.

FIG. 3: UspA2 yield from protein constructs MC-001, MC-002, MC-004,MC-005, MC-006, MC-007, MC-008 and MC-010 evaluated in fermenter; datafrom Table 4.

FIG. 4: Molecular weight distribution of purified MC-005 determined bysedimentation velocity analytical ultracentrifugation. The majority ofprotein is found as a trimer, with a small proportion of a highermolecular weight oligomer that may correspond to dimer of trimer.MW=molecular weight. kDa=kilodalton.

FIG. 5: Molecular weight distribution of purified MC-001 determined bysedimentation velocity analytical ultracentrifugation. Majority ofprotein is found as a trimer.

FIG. 6: Molecular weight distribution of purified MC-001 determined bysedimentation velocity analytical ultracentrifugation. The samplepresents multiple species and is highly polydisperse. The sedimentationcoefficient of the major species detected doesn't correspond to the oneof the trimers normally detected in the other lots.

FIG. 7: Molecular weight distribution of purified MC-001 determined bysedimentation velocity analytical ultracentrifugation. Majority ofprotein is found as a trimer.

FIG. 8: Molecular weight distribution of purified MC-007 determined bysedimentation velocity analytical ultracentrifugation. Majority ofprotein is found as a trimer.

FIG. 9: Far-UV circular dichroism (CD) spectra of UspA2 constructsgiving an indication of protein secondary structures.

FIG. 10: Secondary structures monitoring by circular dichroism (CD)during thermal unfolding of MC-005 (UspA2Δhelix+6His). Visual analysisof the spectra clearly shows that protein loses most of its secondarystructures at 33° C.

FIG. 11: Secondary structures monitoring by circular dichroism (CD)during thermal unfolding of MC-007 (UspA2 full helix+6His). Visualanalysis of the spectra shows that loss of secondary structure is slowercompared to the construct without helix. Structural changes aredetectable upon heating to 33° C., but complete unfolding seems to occurbetween 35° C. and 37° C.

FIG. 12: MALDI spectrum of MC-001 lot opt-01. The mass observed at 57427Da may be coherent with the demethionylated protein, while the peak at57620 Da could correspond to the complete protein.

FIG. 13: MALDI spectrum of MC-011 lot BMP37. The mass observed may becoherent with the demethionylated protein. The two other peaks at +186Da and +366 Da are unidentified.

FIG. 14: Protective efficacy of MC-001 and MC-007 in a mouse model oflung colonization.

FIG. 15: Antibody response directed against UspA2 induced afterintramuscular administration in mice, where PII and PIII indicate,respectively, anti-IgG levels in sera collected at day 28 (post II) andday 42 (post III).

FIG. 16: Bactericidal titers induced by UspA2 against a homologousstrain formulated with different adjuvants (AS01_(E), AS04_(C) andAlPO₄).

FIG. 17: Antibody response directed against UspA2 induced afterintramuscular administration in mice, using different formulations ofantigens and adjuvants.

FIG. 18: Bactericidal titers induced by UspA2 against a homologousstrain, using different formulations of antigens and adjuvants.

FIG. 19: IgG response induced against PD in mice by PD-PEPilA-UspA2vaccine (a trivalent NTHi-M. cat. vaccine), formulated with differentadjuvants.

FIG. 20: IgG response induced against PE in mice by PD-PEPilA-UspA2vaccine (a trivalent NTHi-M. cat. vaccine), formulated with differentadjuvants.

FIG. 21: IgG response induced against PilA in mice by PD-PEPilA-UspA2vaccine (a trivalent NTHi-M. cat. vaccine), formulated with differentadjuvants.

FIG. 22: Immunogenicity of PE in the bivalent PD-PEPilA and trivalentPD-PEPilA-UspA2 formulations with AS01E.

FIG. 23: Immunogenicity of PilA in the bivalent PD-PEPilA and trivalentPE-PilA-UspA2 formulations with AS01E.

FIG. 24: Immunogenicity of PD in the bivalent PD-PEPilA and trivalentPE-PilA-UspA2 formulations with AS01E.

FIG. 25: Effect of the tetravalent PD/PEPilA/UspA2/AS01_(E) vaccineformulation on mouse lungs pre-sensitized with heat inactivated M.cat.—Perivascularitis and peribronchiolitis in PBS immunized mice.

FIG. 26: Effect of the tetravalent PD/PEPilA/UspA2/AS01_(E) vaccineformulation on mouse lungs pre-sensitized with heat inactivated M.cat.—Day 2 post-immunization.

FIG. 27: Effect of the tetravalent PD/PEPilA/UspA2/AS01_(E) vaccineformulation on mouse lungs pre-sensitized with heat inactivated M.cat.—Day 7 post-immunization.

FIG. 28: Effect of the tetravalent PD/PEPilA/UspA2/AS01_(E) vaccineformulation on mouse lungs pre-sensitized with heat inactivated M.cat.—Day 14 post-immunization.

FIG. 29: Effect of the tetravalent PD/PEPilA/UspA2/AS01_(E) vaccineformulation on mouse lungs pre-sensitized with heat inactivated M.cat.—Detailed results.

FIG. 30: Post-vaccination lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IL17. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 31: Post-vaccination lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing TNFα. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 32: Post-vaccination lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IFNγ. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 33: Post-vaccination lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IL13. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 34: Post-challenge lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IL17. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 35: Post-challenge lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing TNFα. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 36: Post-challenge lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IFNα. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

FIG. 37: Post-challenge lung CD4 T cell responses upon M. cat. WCre-stimulation. Lung CD4 cells expressing IL13. Restimulated withheat-inactivated M. cat. whole cells (WC) or medium.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise explained or defined herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. For example, definitions of common terms in molecular biologycan be found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Additionally, numerical limitations given with respect toconcentrations or levels of a substance, such as an antigen may beapproximate. Thus, where a concentration is indicated to be (forexample) approximately 200 pg, it is intended that the concentrationincludes values slightly more or slightly less than (“about” or “˜”) 200pg.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below.

The term “comprises” means “includes”. Thus, unless the context requiresotherwise, the word “comprises,” and variations such as “comprise” and“comprising” will be understood to imply the inclusion of a statedcompound or composition (e.g., nucleic acid, polypeptide, antigen) orstep, or group of compounds or steps, but not to the exclusion of anyother compounds, composition, steps, or groups thereof. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of terms are provided. Additionalterms and explanations are provided in the context of this disclosure.

A “subject” as used herein is a mammal, including humans, non-humanprimates, and non-primate mammals such as members of the rodent genus(including but not limited to mice and rats) and members of the orderLagomorpha (including but not limited to rabbits).

As used herein “UspA2” means Ubiquitous surface protein A2 fromMoraxella catarrhalis. UspA2 may consist of or comprise the amino acidsequence of SEQ ID NO: 1 from ATCC 25238.

(SEQ ID NO: 1) MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEFas well as sequences with at least or exactly 63%, 66%, 70%, 72%, 74%,75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity,over the entire length, to SEQ ID NO: 1. Comparison of 38 sequences ofUspA2 from Moraxella catarrhalis (Table 1, SEQ ID NO: 1-SEQ ID NO: 38)demonstrated approximately 63% to approximately 100% identity to UspA2as set forth in SEQ ID NO. 1.

UspA2 as described in SEQ ID NO: 1 contains a signal peptide (forexample, amino acids 1 to 29 of SEQ ID NO: 1), a laminin binding domain(for example, amino acids 30 to 177 of SEQ ID NO: 1), a fibronectinbinding domain (for example, amino acids 165 to 318 of SEQ ID NO: 1)(Tan et al. JID 192: 1029-38 (2005)), a C3 binding domain (for example,amino acids 30 to 539 of SEQ ID NO: 1 (WO2007/018463), or a fragment ofamino acids 30 to 539 of SEQ ID NO: 1, for example, amino acids 165 to318 of SEQ ID NO: 1 (Hallström T et al. J. Immunol. 186: 3120-3129(2011)), an amphipathic helix (for example, amino acids 519 to 564 ofSEQ ID NO: 1 or amino acids 520-559 of SEQ ID NO:1, identified usingdifferent prediction methods) and a C terminal anchor domain (forexample, amino acids 576 to 630 amino acids of SEQ ID NO: 1 (Brooks etal., Infection & Immunity, 76(11), 5330-5340 (2008)).

UspA2 amino acid differences have been described for various Moraxellacatarrhalis species. See for example, J Bacteriology 181(13):4026-34(1999), Infection and Immunity 76(11):5330-40 (2008) and PLoS One7(9):e45452 (2012).

UspA2 may consist of or comprise an amino acid sequence that differsfrom SEQ ID NO. 1 at any one or more amino acid selected from the groupconsisting of: AA (amino acid) 30 to 298, AA 299 to 302, AA 303 to 333,AA 334 to 339, AA 349, AA 352 to 354, AA 368 to 403, AA 441, AA 451 to471, AA 472, AA474 to 483, AA 487, AA 490, AA 493, AA 529, AA 532 or AA543. UspA2 may consist of or comprise an amino acid sequence thatdiffers from SEQ ID NO: 1 in that it contains at least one amino acidinsertion in comparison to SEQ ID NO. 1. UspA2 may consists of orcomprise an amino acid sequence that differs from SEQ ID NO. 1 at anyone of the amino acid differences in SEQ ID NO: 2 through SEQ ID NO: 38.For example, SEQ ID NO. 1 may contain K instead of Q at amino acid 70, Qinstead of G at amino acid 135 and/or D instead of N at amino acid 216.

TABLE 1UspA2 amino acid sequences from 38 strains of Moraxalla catarrhalis (SEQ IDNO: 1 - SEQ ID NO: 38). Strain UspA2 sequences ATCC 25238MKTMKLLPLKIAVISAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEK (SEQ IDYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQG NO: 1)LADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (630 aa) AmericanMKTMKLLPLKIAVISAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKY 2933LLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEH (SEQ IDDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYT NO: 2)EEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (613 aa) AmericanMKTMKLLPLKIAVTSALIIGLGAASTANAQQQLQTETFLPNFLSNDNYDLTDPFYHNMILGDTA 2912LLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLD (SEQ IDGKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAI NO: 3)KDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (644 aa) AmericanMKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDL 2908QSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPR (SEQ IDKVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQ NO: 4)YLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (591 aa) FinnishMKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQQQQQQQQSRTEIFFPNIFFNENHDELDDAYH 307NIILGDTALLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPV (SEQ IDYQVDYKLDGKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDV NO: 5)TANQQDAIKGLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (687 aa) FinnishMKTMKLLPLKIAVTSAMIVGLGMASTANAQQQKSPKTETFLPNIFFNEYADDLDTLYHNMILGD 353TAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLENGV (SEQ IDQKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREV NO: 6)QNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (683 amino acids) FinnishMKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGN 358TALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGGTIIPLDENGKPVYKLDSIVEQG (SEQ IDKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 7)VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) FinnishMKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQKTKTEVFLPNLFDNDYYDLTDPLYHSMILGD 216TATLFDQQDNSKSQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTQDT (SEQ IDRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAI NO: 8)DALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) Dutch H2MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGN (SEQ IDTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKKGDTIIPLDENGKPVYKLDSIVEQG NO: 9)KIKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIYELVQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) Dutch F10MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENL (SEQ IDQDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYKLDGQEP NO: 10)RRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLIKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF(574 amino acids) NorwegianMKTMKLLPLKIAVTSALIVGLGAASTANAQQQPQTETFFPNIFFNENHDALDDVYHNMILGDTA 1ITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKK (SEQ IDSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQ NO: 11)NNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLIKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) NorwegianMKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGD 13TAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGV (SEQ IDKKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 12)VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) NorwegianMKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDL 20QSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPR (SEQ IDKVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQ NO: 13)YLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (587 amino acids) NorwegianMKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGD 25TAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGV (SEQ IDKKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 14)VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLIKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) NorwegianMKTMKLLPLKIAVTSALIVGLGAASTANAQVRDKSLEDIEALLGKIDISKLEKEKKQQTELQKY 27LLLSQYANVLTMEELNKNVEKNTNSIEALGYEIGWLENDIADLEEGVEELTKNQNTLIEKDEEH (SEQ IDDRLIAQNQADIKTLENNVVEELFNLSDRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYT NO: 15)EEVNKTLEKLITNSVKNTDNIDKNKADIQALENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (616 amino acids) NorwegianMKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQ 36DTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVY (SEQ IDSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNI NO: 16)ENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (676 amino acids) BC5SVMKTMKLLPLKIAVTSALIVGLGAASTANAQNGTSTKLKNLKEYAQYLDNYAQYLDDDIDDL (SEQ IDDKEVGELSQNIAKNQANIKDLNKKLSRDIDSLREDVYDNQYEIVNNQADIEKNQDDIKELE NO: 17)NNVGKELLNLSGRLLDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (629 amino acids) NorwegianMKTMKLLPLKIAVTSAMIVGLGMASTANAQQQRSPKTETFLPNIFFNEYADDLDTLYHNMI 14LGDTAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDK (SEQ IDRLDNGVQKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEE NO: 18)SVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNRIKALENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (683 amino acids) NorwegianMKTMKLLPLKIAVTSAMIVGLGAASTANAQAQSNRSLDQVQALLRGIDETKIKKEIQQSQQ 3PELNKYLTFNQLANALNIEELNNNVQKNTQRLDSAATLYGDLSKTVPKSIKENKESIKENK (SEQ IDESIKENKESIKENKESIKENKESIKENKESITTLTRKSFQNQVDIVRNNASIEDLYAYGQE NO: 19)VAKSIGEIHAYTEEVNKTLENLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTVIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (700 amino acids) FinnishMKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYIETTDPLYHGMILGNTA 414ITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENG (SEQ IDVKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 20)LNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (676 amino acids) JapaneseMKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIV Z7476ENLQDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYK (SEQ IDLDGQEPRRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEE NO: 21)SVQYLNKEVQNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) BelgianMKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKM Z7530NKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLI (SEQ IDEKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAE NO: 22)RIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (613 amino acids) GermanMKTMKLLPLKIAVTSALIVGLGAASTANAQATNKDITLEDVLKSIEEIDPYELRDYIEYPT Z8063AIERFLLLSQYGNTLTLEEFDNDIELLDQDVEDLEESVTELAKNQNSLIEQGEAIKEDLQG (SEQ IDLADFVERQEDKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAKSI NO: 23)GEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALESNVEKGLLELSGHLIDQKADIDNNINNIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (589 amino acids) AmericanMKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMI O12ELGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLD (SEQ IDSIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANE NO: 24)ESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) GreekMKTMKLLPLKIAVTSALIVGLGAASTANAQQQQKTKTEVFLPNLFYNDYIEETDLLYHNMI MC317LGDTAALVDRQNYSNSQLKFYSNDEESVPDSLLFSKMLNNQQLNGFKAGDIIIPVDANGQV (SEQ IDIYQKDTRVEGGKTRTVLSVTTKIATQQDVDSAYSRGIQGKVNDLDDEMNFLNHDITSLYDV NO: 25)TANQQDDIKGLKKGVKDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (650 amino acids) AmericanMKTMKLLPLKIAVTSALIVGLGAVSTTNAQAQSRSLDQIQTKLADLAGKIAAGKNGGGQNN V1122QNNQNDINKYLFLSQYANILTMEELNNNVVKNSSSIETLETDFGWLENDVADLEDGVEELT (SEQ IDKNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSDRLIDQKADIAKNQADIAQNNE NO: 26)SIEELYDFDNEVAEKIGEIHAYTEEVNKTLQDLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (616 amino acids) AmericanMKTMKLLPLKIAVTSALIVGLGTASTANAQVASPANQKIQQKIKKVRKELRQDIKSLRNDI P44DSNTADIGSLNDDVADNQDDILDNQADIAKNQDDIEKNQADIKELDKEVGVLSREIGSLND (SEQ IDDIADNYTDIIDNYTDIIDNQANIAKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDVADN NO: 27)QDDIAKNQADIQTLENNVEEGLLELSGHLLDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQEQTEAIDALNKASSENTQNIAKNSNRIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKVSADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF(668 amino acids) AmericanMKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA V1171IVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ IDRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 28)EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (674 amino acids) AmericanMKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDIDTLKQDQQKM TTA24NKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLI (SEQ IDEKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAE NO: 29)RIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (613 amino acids) AmericanMKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA O5EIVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ IDRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 30)EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (576 amino acids) AmericanMKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMI SP12-6LGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLD (SEQ IDSIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANE NO: 31)ESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) AmericanMKTMKLLPLKIAVTSAMIIGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTA SP12-5ITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENG (SEQ IDVKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 32)LNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (686 amino acids) SwedishMKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEK BC5YLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQG (SEQ IDLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEI NO: 33)HAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (630 amino acids)American MKTMKLLPLKIAVTSALIVGLGAASTANAQAQDRSLEQIQDKLANLVEKIEQAKSQNGQSQ7169 KDINQYLLLSQYANVLTMEELNNNVVKNSSSIETLDNDIAWLNDDLIDLDKEVGVLSRDIG(SEQ ID SLHDDVAQNQADIKTLKNNVVEELFNLSDRLIDQEADIAQNNESIEDLYDFGREVAESIGENO: 34) IHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALENDVGKELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (616 amino acids) FinnishMKTMKLLPLKIAVTSAMIIGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA FIN2344IVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ IDRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 35)EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (614 amino acids) AmericanMKTMKLPPLKIAVTSAMIIGLGAASTANAQTTETFLPNLFDNDYTETTDPLYHGMILGDTA V1118ITQDTQYKFYAENGNEVPDSLFFNKILHDQLLNGFKAGDTIIPLDENGKPVYKLDERTENG (SEQ IDVKRKVYSVTTKTATQADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 36)LNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEECLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (679 amino acids) AmericanMKTMKLLPLKIAVTSALIVGLGAASTANAQETLEEVLESIKQINEQDLQDDIGYNSALDRY V1145LVLSQYGNLLIAKELNENVEKNSNSIAKNSNSIADLEADVGYLAENQNTLIEQNETINQEL (SEQ IDEGITHELESFIAYAHAQDQKNLVNEFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHA NO: 37)YTEEVNKTLENLITNSVKNTDNITKNKADIQALESNVEKELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (724 amino acids)American MKTMKLLPLKIAVTSALIVGLGAASTANAQAQARDRSLEDIQALIGNIDVDKIRSQKQKNPV1156 EIFQYLLLNQLSNTLITDELNNNVIKNTNSIETLDNDIAWLNDDLIDLDKEVGVLSRDIGS(SEQ ID LHDDVAQNQADIKTLENNVVEELFNLSDRLIDQEAEIAQNNESIEDLYDFGREVAESIGEINO: 38) HAHNEAQNETLKDLITNSVKNTDNIDKNKADIQALENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (611 amino acids)

UspA2 may be UspA2 from M. catarrhalis strain ATCC (a US registeredtrademark) 25238™, American 2933. American 2912, American 2908, Finnish307, Finnish 353, Finnish 358, Finnish 216, Dutch H2, Dutch F10,Norwegian 1, Norwegian 13, Norwegian 20, Norwegian 25, Norwegian 27,Norwegian 36, BC5SV, Norwegian 14, Norwegian 3, Finish 414, JapaneseZ7476, Belgium Z7530, German Z8063, American O12E, Greek MC317, AmericanV1122, American P44, American V1171, American TTA24, American 035E,American SP12-6, American SP12-5, Swedish BC5, American 7169, FinnishFIN2344, American V1118, American V1145 or American V1156. UspA2 may beUspA2 as set forth in any of SEQ ID NO: 1-SEQ ID NO: 38. UspA2 may beUspA2 from another source which corresponds to the sequence of UspA2 inany one of SEQ ID NO: 1-SEQ ID NO: 38. Corresponding UspA2 sequences maybe determined by one skilled in the art using various algorithms. Forexample, the Gap program or the Needle program may be used to determineUspA2 sequences corresponding to any one of SEQ ID NO: 1-SEQ ID NO: 38.

UspA2 may be a sequence with at least 95% identity, over the entirelength, to any of SEQ ID NO: 1-SEQ ID NO: 38.

Immunogenic fragments of UspA2 comprise immunogenic fragments of atleast 450 contiguous amino acids of SEQ ID NO: 1, 490 contiguous aminoacids of SEQ ID NO: 1 (for example, the UspA2 fragment of MC-004 orMC-005), 511 contiguous amino acids of SEQ ID NO: 1 (for example, theUspA2 fragment of construct MC-001, MC-002, MC-003 or MC-004), 534contiguous amino acids of SEQ ID NO: 1 (for example, the UspA2 fragmentof MC-009 or MC-011) or 535 contiguous amino acids of SEQ ID NO: 1 (forexample, the UspA2 fragment of MC-007, MC-008 or MC-010). Theimmunogenic fragments may elicit antibodies which can bind SEQ ID NO: 1.

Immunogenic fragments of UspA2 may comprise immunogenic fragments of atleast 450, 490, 511, 534 or 535 contiguous amino acids of any of SEQ IDNO: 1-SEQ ID NO: 38. Immunogenic fragments of UspA2 may compriseimmunogenic fragments of UspA2 from any of SEQ ID NO: 2-SEQ ID NO: 38which correspond to the UspA2 fragment of SEQ ID NO: 1 in any of theUspA2 constructs MC-001, MC-002, MC-003, MC-004, MC-005, MC-006, MC-007,MC-008, MC-009, MC-010 or MC-011. The immunogenic fragments may elicitantibodies which can bind the full length sequence from which thefragment is derived.

Alignments between polypeptides pairs may be calculated by variousprograms. For example, the Needle program from the EMBOSS package (Freesoftware; EMBOSS: The European Molecular Biology Open Software Suite(2000). Trends in Genetics 16(6): 276-277) and the Gap program from theGCG (a US registered trademark) package (Accelrys Inc.) may be used.

The Gap and Needle programs are an implementation of theNeedleman-Wunsch algorithm described in: Needleman, S. B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453. These programs are using frequentlythe BLOSUM62 scoring matrix (Steven Henikoft and Jorja G. Henikoft(1992), “Amino acid substitution matrices from protein blocks”), Proc.Natl. Acad. Sci. USA 89 (Biochemistry): 10915-10919) with gap open andextension penalties of, respectively, 8 and 2. Sometimes, the PAM250scoring matrix (Dayhoft et al., (1978), “A model of evolutionary changesin proteins”, In “Atlas of Protein sequence and structure” 5(3) M. O.Dayhoft (ed.), 345-352, National Biomedical Research Foundation,Washington) is also used.

Scoring matrices are describing by numbers the tendency of each aminoacid to mutate in another, or to be conserved. These numbers aregenerally computed from statistics of mutations observed in faithfulpairwise or multiple alignments, or even in fragments of multiplealignments. Generally, in these tables, if a high positive number isassociated with a pair of identical amino acids, it is indicating thatthis residue has a low tendency for mutation. At the opposite, a highpositive number associated with a pair of different amino acids isindicating a high tendency of mutation between these two. And this iscalled a “conservative substitution”.

Looking at a pairwise alignment, aligned identical residues(“identities”) between the two sequences can be observed. A percentageof identity can be computed by multiplying by 100 (1) the quotientbetween the number of identities and the length of the alignment (forexample, in the Needle program output), or (2) the quotient between thenumber of identities and the length of the longest sequence, or (3) thequotient between the number of identities and the length of the shortestsequence, or (4) the quotient between the number of identities and thenumber of aligned residues (for example, in the Gap program output).

The percentage of identities of Table 8 have been calculated accordingthe definition (3) of the previous paragraph, using the pairwisealignments computed by the Gap software.

As used herein, “adjuvant” means a compound or substance that, whenadministered to a subject in conjunction with a vaccine,immunotherapeutic, or other antigen- or immunogen-containingcomposition, increases or enhances the subject's immune response to theadministered antigen or immunogen (as compared to the immune responsethat would be obtained in the absence of adjuvant). This is to bedistinguished from “adjuvant therapy”, defined by the National CancerInstitute of the United States Institutes of Health in the context ofcancer treatment as additional treatment given after the primarytreatment, to lower the risk that the cancer will recur.

The invention further provides proteins of formula (I) containingconservative amino acid substitutions. For example, the proteins offormula (I) may contain a conservative substitution of any amino acidfrom UspA2 of Moraxella catarrahlis as described in any of the sequencesset forth herein (for example, any UspA2 sequence set forth in SEQ IDNO. 1-SEQ ID NO. 38).

As used herein “signal peptide” refers to a short (less than 60 aminoacids, for example, 3 to 60 amino acids) polypeptide present onprecursor proteins (typically at the N terminus), and which is typicallyabsent from the mature protein. The signal peptide (sp) is typicallyrich in hydrophobic amino acids. The signal peptide directs thetransport and/or secretion of the translated protein through themembrane. Signal peptides may also be called targeting signals, transitpeptides, localization signals, or signal sequences. For example, thesignal sequence may be a co-translational or post-translational signalpeptide.

A heterologous signal peptide may be cleaved from a protein construct bysignal peptide peptidases during or after protein transportation orsecretion. For example, the signal peptide peptidase is signal peptidepeptidase I. A “heterologous” signal peptide is one which is notassociated with the protein as it exists in nature.

As used herein “treatment” means the prevention of occurrence ofsymptoms of the condition or disease in a subject, the prevention ofrecurrence of symptoms of the condition or disease in a subject, thedelay of recurrence of symptoms of the condition or disease in asubject, the decrease in severity or frequency of symptoms of thecondition or disease in a subject, slowing or eliminating theprogression of the condition and the partial or total elimination ofsymptoms of the disease or condition in a subject.

As used herein, “optionally” means that the subsequently describedevent(s) may or may not occur, and includes both event(s) that occur andevents that do not occur.

Otitis media is a major cause of morbidity in 80% of all children lessthan 3 years of age. (Expert Rev. Vaccines 5:517-534 (2006)). More than90% of children develop otitis media before age 7 (Current Opinion inInvestigational Drugs 4:953-958 (2003)). In 2000, there were 16 millionvisits made to office-based physicians for otitis media in the UnitedStates and approximately 13 million antibacterial prescriptionsdispensed. (Pediatrics 113:1451-1465 (2004)). In European countries, thereported acute otitis media rates range between 0.125 to 1.24 perchild-year. (Expert Review of Vaccines 8:1479-1500 (2009)). Otitis mediais a costly infection and the most common reason children receiveantibiotics. (Current Infectious Disease Reports 11:177-182 (2009)).Bacteria are responsible for approximately 70% of cases of acute otitismedia, with Streptococcus pneumoniae, non-typeable Haemophilusinfluenzae (NTHi), and Moraxella catarrhalis predominating as thecausative agents (Expert Review of Vaccines 5:517-534 (2006)). A subsetof children experience recurrent and chronic otitis media and theseotitis prone children have protracted middle-ear effusions that areassociated with hearing loss and delays in speech and languagedevelopment. (Current Infectious Disease Reports 11:177-182 (2009)).Recent antibiotic pressure and vaccination with the pneumococcalconjugate vaccine have resulted in the emergence of13-lactamase-producing Haemophilus influenzae and Moraxella catarrhalisas the leading organisms causing acute otitis media in North America,followed by Streptococcus pneumoniae (Pediatr Clin N Am 60 (2013)391-407).

Since otitis media is a multifactorial disease, the feasibility ofpreventing otitis media using a vaccination strategy has beenquestioned. (Current Infectious Disease Reports 11:177-182 (2009)).

The chinchilla model is a robust and validated animal model of otitismedia and its prevention (Expert Review of Vaccines 8:1063-1082 (2009)).While the chinchilla model may mimic the natural course of humaninfection, others have suggested that results in the chinchilla modelmay vary from one laboratory to the next. (Current Opinion inInvestigational Drugs 4:953-958 (2003)).

Various other rodents have also been used for the induction of otitismedia and are summarized in Vaccine 26:1501-1524 (2008). The murineanimal model is often studied in otitis media research.

The presence of bactericidal antibody is associated with protection fromotitis media due to non-typeable H. influenzae. (Current Opinion inInfectious Disease 16:129-134 (2003)). However, an immune response neednot be bactericidal to be effective against NTHi. Antibodies that merelyreact with NTHi surface adhesins can reduce or eliminate otitis media inthe chinchilla. (Current Opinion in Investigational Drugs 4:953-958(2003)).

Chronic obstructive pulmonary disease is a chronic inflammatory diseaseof the lungs and a major cause of morbidity and mortality worldwide.Approximately one in 20 deaths in 2005 in the US had COPD as theunderlying cause. (Drugs and Aging 26:985-999 (2009)). It is projectedthat in 2020 COPD will rise to the fifth leading cause of disabilityadjusted life years, chronic invalidating diseases, and to the thirdmost important cause of mortality (Lancet 349:1498-1504 (1997)).

The course of COPD is characterized by progressive worsening of airflowlimitation and a decline in pulmonary function. COPD may be complicatedby frequent and recurrent acute exacerbations (AE), which are associatedwith enormous health care expenditure and high morbidity. (Proceedingsof the American Thoracic Society 4:554-564 (2007)). One study suggeststhat approximately 50% of acute exacerbations of symptoms in COPD arecaused by non-typeable Haemophilus influenzae, Moraxella catarrhalis,Streptococcus pneumoniae, and Pseudomonas aeruginosa. (Drugs and Aging26:985-999 (2009)). Haemophilus influenzae (H. influenzae) is found in20-30% of exacerbations of COPD; Streptococcus pneumoniae, in 10-15% ofexacerbations of COPD; and Moraxella catarrhalis, in 10-15% ofexacerbations of COPD. (New England Journal of Medicine 359:2355-2365(2008)). Haemophilus influenzae, Streptococcus pneumoniae, and Moraxellacatarrhalis have been shown to be the primary pathogens in acuteexacerbations of bronchitis in Hong Kong, South Korea, and thePhillipines, while Klebsiella spp., Pseudomonas aeruginosa andAcinetobacter spp. constitute a large proportion of pathogens in otherAsian countries/regions including Indonesia, Thailand, Malaysia andTaiwan (Respirology, (2011) 16, 532-539;doi:10.1111/j.1440.1843.2011.01943.x). In Bangladesh, 20% of patientswith COPD showed positive sputum culture for Pseudomonas, Klebsiella,Streptococcus pneumoniae and Haemophilus influenzae, while 65% ofpatients with AECOPD (acute exacerbation of COPD) showed positivecultures for Pseudomonas, Klebsiella, Acinetobacter, Enterobacter,Moraxella catarrhalis and combinations thereof. (Mymensingh MedicalJournal 19:576-585 (2010)). However, it has been suggested that the twomost important measures to prevent COPD exacerbation are activeimmunizations and chronic maintenance of pharmacotherapy. (Proceedingsof the American Thoracic Society 4:554-564 (2007)).

Community-acquired pneumonia (CAP) has been described as the leadingcause of death from infectious disease and the six-ranked cause of deathoverall in the United States. Moraxella catarrhalis is one of thepathogens associated with CAP in North America (Clin Chest Med 26 (2005)37-55) and is one of the pathogens associated with moderate to severecommunity acquired pneumonia in Japan (J Infect Chemother. 2014 Nov. 20.pii: S1341-321X(14)00396-1. doi: 10.1016/j.jiac.2014.11.006. [Epub aheadof print]).

There is a need for effective vaccines against M. catarrhalis.

The present invention relates to proteins of formula (I).A-(R₁)_(m)—(B)_(n)  (formula I)

wherein:

A is UspA2 from Moraxella catarrhalis or an immunogenic fragmentthereof;

R₁ is an amino acid;

m is 0 or 2;

B is histidine; and

n is 0, 1, 2, 3, 4, 5 or 6.

In one particular embodiment, R₁ and m are defined wherein (R₁)_(m) isAS (alanine serine). In another embodiment, R₁ is non-native aminoacids.

In one embodiment, the proteins of formula (I) and proteins of theinvention are defined wherein m is 0. In one embodiment, when m is 0, nis 2. In another embodiment of the invention, when m is 0, n is not 0.

In one embodiment, m is 2.

In one particular embodiment, n is selected from the group consisting of1, 2 and 6. In another embodiment, n is selected from the groupconsisting of 2 and 6. In one particular embodiment, n is 2. In anotherembodiment, n is 6.

In one embodiment, n is selected from the group consisting of 0, 1, 2,and 6, or any subset thereof.

In one embodiment n is 0. In another embodiment, when n is 0, m is 2.

In one embodiment, n is 1. In one embodiment, n is 3. In one embodiment,n is 4. In one embodiment, n is 5.

In one embodiment, the proteins of formula (I) further contain amethionine (M) at the amino terminus; a protein with the followingformula: methionine-A-(R₁)_(m)—(B)_(n). These are included withinproteins of the invention. In one particular embodiment, when m is 0 andn is 0, the proteins of formula (I) and proteins of the invention arenon-native proteins.

In one embodiment, the proteins of formula (I) and proteins of theinvention are non-native proteins.

In one embodiment, the proteins of formula (I) are defined wherein A isUspA2 from M. catarrhalis. In another embodiment, the proteins offormula (I) are defined wherein A is UspA2 as set forth in an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37 and SEQ ID NO: 38 or any subset of SEQ ID NO: 1through SEQ ID NO:38. In another embodiment, the proteins of formula (I)are defined wherein A is UspA2, wherein UspA2 is at least 63%, 66%, 70%,72%, 74%, 75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical, over the entire length, to SEQ ID NO: 1. In anotherembodiment, the proteins of formula (I) are defined wherein A is UspA2,wherein UspA2 is approximately 75% to 100% identical to the UspA2 aminoacid sequence set forth in SEQ ID NO: 1. In another embodiment, A isUspA2 wherein UspA2 is approximately 90% to 100% identical to the UspA2amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, Ais UspA2 wherein UspA2 is at least 95% identical to the UspA2 amino acidsequence set forth in SEQ ID NO: 1. In another embodiment, the proteinsof formula (I) are defined wherein A is UspA2, wherein UspA2 isapproximately 75% to 100% identical to the UspA2 amino acid sequence setforth in any one of SEQ ID NO: 1-SEQ ID NO: 38. In another embodiment, Ais UspA2 wherein UspA2 is approximately 90% to 100% identical to theUspA2 amino acid sequence set forth in any one of SEQ ID NO: 1-SEQ IDNO: 38. In additional embodiment, A is UspA2 wherein UspA2 is at least95% identical to UspA2 as set forth in any of SEQ ID NO: 1-SEQ ID NO:38. In a particular embodiment, A is UspA2 having the amino acidsequence set forth in SEQ ID NO. 1.

In another embodiment, the proteins of formula (I) are defined wherein Ais an immunogenic fragment of UspA2 from M. catarrhalis. In anotherembodiment, A is an immunogenic fragment of UspA2 wherein UspA2 has anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 or any subset of SEQID NO: 1 through SEQ ID NO: 38. In another embodiment, A is animmunogenic fragment of UspA2, wherein UspA2 is approximately 75% to100% identical to the amino acid sequence set forth in SEQ ID NO: 1. Inanother embodiment, A is an immunogenic fragment of UspA2, wherein UspA2is approximately 90% to 100% identical to SEQ ID NO. 1. In an anotherembodiment, A is an immunogenic fragment of UspA2, wherein UspA2 is atleast 95% identical to SEQ ID NO: 1. In another embodiment, A is animmunogenic fragment of UspA2, wherein UspA2 is approximately 75% to100% identical to the amino acid sequence set forth in any one of SEQ IDNO: 1-SEQ ID NO: 38. In another embodiment, A is an immunogenic fragmentof UspA2, wherein UspA2 is approximately 90% to 100% identical to anyone of SEQ ID NO: 1-SEQ ID NO: 38. In an additional embodiment, A is animmunogenic fragment of UspA2, wherein UspA2 is at least 95% identicalto any of SEQ ID NO: 1-SEQ ID NO: 38. In a particular embodiment, A isan immunogenic fragment of UspA2 wherein UspA2 has the amino acidsequence set forth in SEQ ID NO: 1.

In another embodiment, A is an immunogenic fragment of UspA2 from M.catarrhalis selected from the group consisting of amino acids 30-540 ofSEQ ID NO. 1 (SEQ ID NO: 39), amino acids 31-540 of SEQ ID NO: 1 (SEQ IDNO: 40), amino acids 30-519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids30-564 of SEQ ID NO: 1 (SEQ ID NO: 42) and amino acids 31-564 of SEQ IDNO: 1 (SEQ ID NO: 43). More specifically, in one embodiment, A is SEQ IDNO: 43, amino acids 31-564 of SEQ ID NO: 1. In an additional embodiment,A is SEQ ID NO: 42, amino acids 30-564 of SEQ ID NO: 1. In anotherembodiment, A is an immunogenic fragment of UspA2 from M. catarrhalisselected from the group consisting of amino acids 30-540 of SEQ ID NO. 1(SEQ ID NO:39), amino acids 31-540 of SEQ ID NO. 1 (SEQ ID NO: 40) andamino acids 30-519 of SEQ ID NO. 1 (SEQ ID NO: 41). In anotherembodiment, A is an immunogenic fragment of UspA2 with at least 52%(American 2908), 55% (Norwegian 25), 57% (Japanese Z7476), 62% (FinnishFIN2344), 64% (American 2912), 69% (American P44), 73% (American 7169),76% (Norwegian 27), 81% (American V1145), 88% (German Z8063) or 100%(Swedish BC5) identity to SEQ ID NO. 39. In another embodiment, A is animmunogenic fragment of UspA2 with at least 52% (American 2908), 57%(Dutch F10), 62% (American 2933), 65% (Greek MC317), 67% (AmericanV1122), 70% (American P44), 73% (American 7169), 76% (Norwegian 3), 81%(German Z8063), 100% (Swedish BC5) identical to SEQ ID NO. 43.

In another embodiment, A is an immunogenic fragment of UspA2 from M.catarrhalis from SEQ ID NO: 2 through SEQ ID NO: 38 where the fragmentcomprises the amino acids that align with amino acids 30-540 of SEQ IDNO. 1 (SEQ ID NO: 39), amino acids 31-540 of SEQ ID NO: 1 (SEQ ID NO:40), amino acids 30-519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids30-564 of SEQ ID NO: 1 (SEQ ID NO: 42) or amino acids 31-564 of SEQ IDNO: 1 (SEQ ID NO: 43). In one embodiment, the Gap program (from the GCGpackage), or the Needle program (from the EMBOSS package), implementingthe Needleman-Wunsch algorithm, may be used to align the sequences.

UspA2- SEQ ID NO: 1MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEFAmino acides 30-540 of UspA2 from SEQ ID NO: 1,  SEQ ID NO: 39QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKAmino acids 31-540 of UspA2 from SEQ ID NO: 1, SEQ ID NO: 40AKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKAmino acids 30-519 of UspA2 from SEQ ID NO: 1, SEQ ID NO: 41QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSAmino acids 30-564 of UspA2 from SEQ ID NO: 1, SEQ ID NO: 42QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA Amino acids 31-564 of UspA2 from SEQ ID NO: 1,SEQ ID NO: 43 AKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA

In another embodiment, A is an immunogenic fragment of UspA2 from M.catarrhalis that differs from SEQ ID NO: 1 in one or more of thefollowing amino acids: AA (amino acid) 30 to 298, AA 299 to 302, AA 303to 333, AA 334 to 339, AA 349, AA 352 to 354, AA 368 to 403, AA 441, AA451 to 471, AA 472, AA 474 to 483, AA 487, AA 490, AA 493, AA 529, AA532 or AA 543. In another embodiment, A is an immunogenic fragment ofUspA2 from M. catarrhalis that differs from SEQ ID NO: 1 in that itcontains at least one amino acid insertion in comparison to SEQ ID NO.1.

In another embodiment, A is an immunogenic fragment of UspA2 thatcontains a laminin binding domain and a fibronectin binding domain.

In an additional embodiment, A is an immunogenic fragment of UspA2 thatcontains a laminin binding domain, a fibronectin binding domain and a C3binding domain.

In a further embodiment, A is an immunogenic fragment of UspA2 thatcontains a laminin binding domain, a fibronectin binding domain, a C3binding domain and an amphipathic helix.

The laminin binding domain, fibronectin binding domain, C3 bindingdomain or amphipathic helix may be as defined for SEQ ID NO: 1 or may bethe corresponding sequence in any one of SEQ ID NO: 2 through SEQ ID NO:38.

Proteins of formula (I) and proteins of the invention are useful asimmunogens in subjects such as mammals, particularly humans. Inparticular, the proteins of formula (I) and proteins of the inventionare useful in inducing an immune response against M. catarrhalis insubjects, particularly humans. The proteins of formula (I) and proteinsof the invention are useful in the treatment or prevention of M.catarrhalis infection or disease. More specifically, the proteins offormula (I) and proteins of the invention are useful in the treatment orprevention of otitis media and/or COPD and/or AECOPD and/or pneumonia.

The present invention relates to immunogenic compositions comprisingUspA2 from M. catarrhalis or an immunogenic fragment thereof. Thepresent invention also relates to vaccines comprising such immunogeniccompositions and therapeutic uses of the same. Immunogenic compositionsand vaccines of the present invention are useful in the treatment orprevention of M. catarrhalis infection or disease. More specifically,immunogenic compositions and vaccines described herein are useful in thetreatment or prevention of otitis media and/or COPD and/or AECOPD and/orpneumonia.

In one embodiment, the immunogenic composition comprises UspA2 from M.catarrhalis. UspA2 may be any one of SEQ ID NO: 1 through SEQ ID NO: 38or a UspA2 sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to any one of SEQ ID NO: 1 through SEQ ID NO: 38. UspA2may also be a UspA2 sequence at least 63% (American 2908), 66% (JapaneseZ7476), 70% (Dutch F10), 72% (Finnish 358), 74% (American P44), 77%(Finnish 307), 80% (Norwegian 3), 84% (American V1145), 90% (GermanZ8063) or 100% (Swedish BC5) identical to that of SEQ ID NO. 1.

In another embodiment, the immunogenic composition comprises animmunogenic fragment of UspA2. The immunogenic fragment of UspA2 may beSEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ IDNO.43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%sequence identity to any one of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42 or SEQ ID NO.43. The immunogenic fragment of UspA2 maybe a UspA2 sequence at least 52% (American 2908), 55% (Norwegian 25),57% (Japanese Z7476), 62% (Finnish FIN2344), 64% (American 2912), 69%(American P44), 73% (American 7169), 76% (Norwegian 27), 81% (AmericanV1145), 88% (German Z8063) or 100% (Swedish BC5) identical to SEQ ID NO.39. The immunogenic fragment of UspA2 may also be a UspA2 sequence atleast 52% (American 2908), 57% (Dutch F10), 62% (American 2933), 65%(Greek MC317), 67% (American V1122), 70% (American P44), 73% (American7169), 76% (Norwegian 3), 81% (German Z8063), 100% (Swedish BC5)identical to SEQ ID NO. 43. Amino acid differences have been describedin UspA2 from various Moraxella catarrhalis species.

UspA2 contains a laminin binding domain (for example, amino acids 30-177of SEQ ID NO: 1, SEQ ID NO: 44). In one embodiment, the fragment ofUspA2 comprises the laminin binding region of SEQ ID NO: 1. In anadditional embodiment, the fragment of UspA2 comprises the lamininbinding region of any one of SEQ ID NO: 2-SEQ ID NO: 38.

Amino acids 30-177 of SEQ ID NO: 1, SEQ ID NO: 44:QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIE D.

UspA2 contains a fibronectin binding domain (for example, amino acids165-318 of SEQ ID NO: 1, SEQ ID NO: 45). In one embodiment, the fragmentof UspA2 comprises the fibronectin binding region of SEQ ID NO: 1. In anadditional embodiment, the fragment of UspA2 comprises the fibronectinbinding region of any one of SEQ ID NO: 2-SEQ ID NO:38. The fibronectinbinding domain of SEQ ID NO: 45 also has C3 binding properties.

Amino acids 165-318 of SEQ ID NO: 1, SEQ ID NO: 45: KDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQK.

UspA2 contains a complement component 3 (C3) binding domain (forexample, amino acids 30-539 of SEQ ID NO: 1, SEQ ID NO: 46, or aminoacids 165-318 of SEQ ID NO: 1, SEQ ID NO: 45). In one embodiment, thefragment of UspA2 comprises the C3 binding region of SEQ ID NO: 1. In anadditional embodiment, the fragment of UspA2 comprises a C3 bindingdomain of any one of SEQ ID NO: 2-SEQ ID NO: 38.

Amino acids of 30-539 of SEQ ID NO: 1, SEQ ID NO: 46:QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDT

UspA2 contains an amphipathic helix (for example, amino acids 519-564 ofSEQ ID NO: 1 or or amino acids 520-559 of SEQ ID NO:1). In oneembodiment, the fragment of UspA2 comprises amino acids 519-564 of SEQID NO: 1. In another embodiment, the fragment of UspA2 comprises aminoacids 520-559 of SEQ ID NO:1. In an additional embodiment, the fragmentof UspA2 comprises an amphipathic helix of any one of SEQ ID NO: 2-SEQID NO:38.

In one embodiment, the immunogenic composition comprises a protein offormula (I) wherein A is an immunogenic fragment of UspA2 that comprisesa laminin binding domain and a fibronectin binding domain.

In an additional embodiment, the immunogenic composition comprises aprotein of formula (I) wherein A is an immunogenic fragment of UspA2that comprises a laminin binding domain, a fibronectin binding domainand a C3 binding domain.

In a further embodiment, the immunogenic composition comprises a proteinof formula (I) wherein A is an immunogenic fragment of UspA2 thatcomprises a laminin binding domain, a fibronectin binding domain, a C3binding domain and an amphipathic helix.

In another embodiment, the immunogenic composition comprises a proteinas defined by formula (I). The immunogenic composition may contain, forexample, a protein of formula (I) with an additional methionine at theamino terminus.

In one embodiment, the present immunogenic compositions may beadministered with other antigens. For example, the present immunogeniccomposition may be administered with antigens from H. influenzae. Forexample, the protein of formula (I) may be administered with Protein D(PD) from H. influenzae. Protein D may be as described in WO91/18926.The present immunogenic composition may be administered with Protein E(PE) and Pilin A (PilA) from H. Influenzae. Protein E and Pilin A may beas described in WO2012/139225; the contents of which are incorporatedherein by reference. Protein E and Pilin A may be presented as a fusionprotein.

In another embodiment, the immunogenic compositions of the invention maybe administered with additional antigens from other bacterial speciesalso known to cause otitis media, COPD, AECOPD or pneumonia.

The amount of the immunogenic composition which is required to achievethe desired therapeutic or biological effect will depend on a number offactors such as the use for which it is intended, the means ofadministration, the recipient and the type and severity of the conditionbeing treated, and will be ultimately at the discretion of the attendantphysician or veterinarian. In general, a typical dose for the treatmentof a condition caused in whole or in part by M. catarrhalis in a human,for instance, may be expected to lie in the range of from about 0.001mg-0.120 mg. More specifically, a typical dose for the treatment of acondition caused wholly or in part by M. catarrhalis in a human may liein the range of from about 0.003 mg to about 0.03 mg of protein. Thepresent invention provides an immunogenic composition comprising aprotein of formula (I) or a protein of the invention for use in thetreatment or prevention of a condition or disease caused wholly or inpart by M. catarrhalis. The immunogenic composition may containadditional antigens; a typical dose for the treatment of a conditioncaused wholly or in part by H. influenzae in a human may lie in therange of from about 0.005 mg to about 0.05 mg for each additionalantigen. This dose may be administered as a single unit dose. Severalseparate unit doses may also be administered. For example, separate unitdoses may be administered as separate priming doses within the firstyear of life or as separate booster doses given at regular intervals(for example, every 1, 5 or 10 years). The present invention alsoprovides an immunogenic composition comprising a protein of formula (I)or a protein of the invention for use in the treatment or prevention ofa condition or disease caused wholly or in part by Moraxella catarrhalisin combination with at least one antigen from Haemophilus influenzae.

Formulations comprising the immunogenic compositions of the inventionmay be adapted for administration by an appropriate route, for example,by the intramuscular, sublingual, transcutaneous, intradermal orintranasal route. Such formulations may be prepared by any method knownin the art.

The immunogenic compositions of the present invention may additionallycomprise an adjuvant. When the term “adjuvant” is used in thisspecification, it refers to a substance that is administered inconjunction with the immunogenic composition to boost the patient'simmune response to the immunogenic component of the composition.

Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel or aluminum phosphate or alum, but may also be a salt of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedsaccharides, or polyphosphazenes. In one embodiment, the protein may beadsorbed onto aluminium phosphate. In another embodiment, the proteinmay be adsorbed onto aluminium hydroxide. In a third embodiment, alummay be used as an adjuvant.

Suitable adjuvant systems which promote a predominantly Th1 responseinclude: non-toxic derivatives of lipid A, Monophosphoryl lipid A (MPL)or a derivative thereof, particularly 3-de-O-acylated monophosphoryllipid A (3D-MPL) (for its preparation see GB 2220211 A); and acombination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A, together with either an aluminum salt (forinstance aluminum phosphate or aluminum hydroxide) or an oil-in-wateremulsion. In such combinations, antigen and 3D-MPL are contained in thesame particulate structures, allowing for more efficient delivery ofantigenic and immunostimulatory signals. Studies have shown that 3D-MPLis able to further enhance the immunogenicity of an alum-adsorbedantigen (Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1).

AS01 is an Adjuvant System containing MPL (3-O-desacyl-4′-monophosphoryllipid A), QS21 ((Quillaja saponaria Molina, fraction 21) Antigenics, NewYork, N.Y., USA) and liposomes. AS01B is an Adjuvant System containingMPL, QS21 and liposomes (50 μg MPL and 50 μg QS21). AS01E is an AdjuvantSystem containing MPL, QS21 and liposomes (25 μg MPL and 25 μg QS21). Inone embodiment, the immunogenic composition or vaccine comprises AS01.In another embodiment, the immunogenic composition or vaccine comprisesAS01B or AS01E. In a particular embodiment, the immunogenic compositionor vaccine comprises AS01E.

AS02 is an Adjuvant System containing MPL and QS21 in an oil/wateremulsion. AS02V is an Adjuvant System containing MPL and QS21 in anoil/water emulsion (50 μg MPL and 50 μg Q521).

AS03 is an Adjuvant System containing α-Tocopherol and squalene in anoil/water (o/w) emulsion. AS03_(A) is an Adjuvant System containingα-Tocopherol and squalene in an o/w emulsion (11.86 mg tocopherol).AS03_(B) is an Adjuvant System containing α-Tocopherol and squalene inan o/w emulsion (5.93 mg tocopherol). AS03c is an Adjuvant Systemcontaining α-Tocopherol and squalene in an o/w emulsion (2.97 mgtocopherol). In one embodiment, the immunogenic composition or vaccinecomprises AS03.

AS04 is an Adjuvant System containing MPL (50 μg MPL) adsorbed on analuminum salt (500 μg Al³⁺). In one embodiment, the immunogeniccomposition or vaccine comprises AS04.

A system involving the use of QS21 and 3D-MPL is disclosed in WO94/00153. A composition wherein the QS21 is quenched with cholesterol isdisclosed in WO 96/33739. An additional adjuvant formulation involvingQS21, 3D-MPL and tocopherol in an oil in water emulsion is described inWO 95/17210. In one embodiment the immunogenic composition additionallycomprises a saponin, which may be QS21. The formulation may alsocomprise an oil in water emulsion and tocopherol (WO 95/17210).Unmethylated CpG containing oligonucleotides (WO 96/02555) and otherimmunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are alsopreferential inducers of a TH1 response and are suitable for use in thepresent invention.

Additional adjuvants are those selected from the group of metal salts,oil in water emulsions, Toll like receptor agonists, (in particular Tolllike receptor 2 agonist, Toll like receptor 3 agonist, Toll likereceptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8agonist and Toll like receptor 9 agonist), saponins or combinationsthereof.

The present invention provides a process for preparing an immunogeniccomposition comprising combining a protein of formula (I) or a proteinof the invention with an adjuvant.

The present invention further provides a vaccine containing animmunogenic composition of the invention and a pharmaceuticallyacceptable adjuvant.

Possible excipients include arginine, pluronic acid and/or polysorbate.In a preferred embodiment, polysorbate 80 (for example, TWEEN (a USregistered trademark) 80) is used. In a further embodiment, a finalconcentration of about 0.03% to about 0.06% is used. Specifically, afinal concentration of about 0.03%, 0.04%, 0.05% or 0.06% polysorbate 80(w/v) may be used.

The present invention provides a process for preparing an immunogeniccomposition or vaccine comprising combining a protein of formula (I) orprotein of the invention with a pharmaceutically acceptable excipient.

The present invention also provides nucleic acids encoding the proteinsof the invention. The term “nucleic acid” refers to a polymeric form ofnucleotides. Nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either ribonucleotides or deoxyribonucleotides. Theterm includes single and double forms of DNA. The nucleic acids arepreferably substantially free from other nucleic acids.

The present invention provides a process of producing nucleic acids ofthe invention. Nucleic acids of the invention may be prepared by methodsknown by those skilled in the art. For example, the nucleic acids of theinvention may be synthesized in part or in whole. The nucleic acids maybe prepared by digesting longer amino acids or joining shorter aminoacids.

The present invention provides a method for the treatment or preventionof otitis media. The method comprises administering to a subject in needthereof a therapeutically effective amount of a protein of formula (I)or a protein of the invention.

The present invention provides a method for the treatment or preventionof exacerbations in chronic obstructive pulmonary disease. Theexacerbation of COPD may be an acute exacerbation. The method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the protein of formula (I) or a protein of the invention.

The present invention provides a method for the treatment or preventionof pneumonia. The method comprises administering to a subject in needthereof a therapeutically effective amount of the protein of formula (I)or a protein of the invention.

The present invention provides a pharmaceutical composition comprising aprotein of formula (I) or a protein of the invention for use in thetreatment or prevention of a condition or disease caused wholly or inpart by Moraxella catarrhalis. Pharmaceutical compositions may furthercomprise a pharmaceutically acceptable adjuvant.

The present invention provides use of (a) proteins of formula (I) andproteins of the invention, (b) an immunogenic composition comprising aprotein of formula (I) or protein of the invention or (c) a vaccinecomprising (c1) a protein of formula (I) or protein of the invention or(c2) immunogenic composition comprising a protein of formula (I) orprotein of the invention for the manufacture of a medicament fortreating or preventing M. catarrhalis infection or disease.

The present invention provides use of (a) proteins of formula (I) andproteins of the invention, (b) an immunogenic composition comprising aprotein of formula (I) or protein of the invention or (c) a vaccinecomprising (c1) a protein of formula (I) or protein of the invention or(c2) immunogenic composition comprising a protein of formula (I) orprotein of the invention for the manufacture of a medicament fortreating or preventing otitis media.

The present invention provides use of (a) proteins of formula (I) andproteins of the invention, (b) an immunogenic composition comprising aprotein of formula (I) or protein of the invention or (c) a vaccinecomprising (c1) a protein of formula (I) or protein of the invention or(c2) immunogenic composition comprising a protein of formula (I) orprotein of the invention for the manufacture of a medicament fortreating or preventing acute exacerbations of chronic obstructivepulmonary disease (AECOPD).

The present invention provides a use of (a) proteins of formula (I) andproteins of the invention, (b) an immunogenic composition comprising aprotein of formula (I) or protein of the invention or (c) a vaccinecomprising (c1) a protein of formula (I) or protein of the invention or(c2) immunogenic composition comprising a protein of formula (I) orprotein of the invention for the manufacture of a medicament fortreating or preventing pneumonia.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way.

In the examples, the following terms have the designated meaning:

6×his=six histidines;

xg=centrifugal force (number gravities)

AS=alanine serine

BSA=bovine serum albumin;

° C.=degrees Celsius;

CaCl₂)=calcium chloride;

CD=circular dichroism;

CHCl₃=chloroform;

CH₃CN=acetonitrile;

CO₂=carbon dioxide;

Da=dalton;

DNA=deoxyribonucleic acid;

DO=dissolved oxygen;

DSC=differential scanning calorimetry;

EDTA=ethylenediaminetetraacetic acid;

h=hour;

H₂O=water;

H₂O₂=hydrogen peroxide;

HCDI=high cell density induction;

HCl=hydrogen chloride;

His=his=histidine;

IMAC=immobilized metal affinity chromatography;

IPTG=isopropyl β-D-1-thiogalactopyranoside;

kVolts=kilovolts

L=liter;

LB=Luria-Bertani;

LCD=low cell density induction;

MeOH=methanol;

ml=milliliter;

NaCl=sodium chloride;

RPM=rpm=revolutions per minute;

min=minute;

mM=millimolar;

μg=microgram;

μL=microliter;

MW=molecular weight;

m/z=mass/charge;

NaCl=sodium chloride;

NaPO₄=sodium phosphate;

ng=nanogram;

NH₄OH=ammonium hydroxide;

nm=nanometer;

O.D.=optical density;

PBS=phosphate buffered saline;

PCR=polymerase chain reaction;

psi=pounds per square inch;

PVDF=polyvinylidence diluoride;

SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis;

TFA=trifluoroacetic acid

Tm=melting point;

Tm₁=first melting point;

Tm₂=second melting point;

w/v=weight/volume.

EXAMPLES Example 1: Protein Constructs

Protein constructs were produced with different fragments of UspA2 withand without additional amino acids. The following table describesprotein constructs made.

TABLE 2 Protein constructs containing UspA2 protein. Construct IDDescription N-terminal---------------------------------------------------C-Terminal MC-001UspA2 + 1/2 helix + 6His UspA2 fragment ASHHHHHH (A.A.: 30-540 of SEQ IDNO: 1, SEQ ID NO: 47) A.A. 30 540 541      548 MC-002 UspA2 + 1/2 helixUspA2 fragment (A.A.: 30-540 of SEQ ID NO: 1, SEQ ID NO: 47) A.A. 30 540MC-003 UspA2 + 1/2 helix + 1His UspA2 fragment (A.A.: 30-540 of SEQ IDNO:. 1, SEQ ID NO: 47) H A.A. 30 540 541 MC-004 UspA2 + 1/2 helix + 2HisUspA2 fragment HH (A.A.: 30-540 of SEQ ID NO: 1, SEQ ID NO: 47) A.A. 30540 541  542 MC-005 UspA2 Δhelix + 6His UspA2 fragment ASHHHHHH (A.A.:30-519 of SEQ ID NO: 1, SEQ ID NO: 48) A.A. 30 519 520      527 MC-006UspA2 Δhelix UspA2 fragment (A.A.: 30-519 of SEQ ID NO: 1, SEQ ID NO:48) A.A. 30 519 MC-007 UspA2 + helix + 6His UspA2 fragment ASHHHHHH(A.A.: 30-564 of SEQ ID NO: 1, SEQ ID NO: 49) A.A. 30 564 565   572MC-008 UspA2 + helix + 2His UspA2 fragment HH (A.A.: 30-564 of SEQ IDNO: 1, SEQ ID NO: 49) A.A. 30      564 565 566 MC-009 UspA2 + helix +2His ΔQ UspA2 fragment HH (A.A.: 31-564 of SEQ ID NO: 1, SEQ ID NO: 50)A.A. 31 564 565 566 MC-010 UspA2 + helix UspA2 fragment (A.A.: 30-564 ofSEQ ID NO: 1, SEQ ID NO: 49) A.A. 30 564 MC-011 UspA2 + 1/2 helix + 6HisΔQ UspA2 fragment ASHHHHHH (A.A.: 31-540 of SEQ ID NO: 1, SEQ ID NO: 51)A.A. 31 540 541   548 A.A. = amino acid

The DNA and amino acid sequences for each protein constructs listed inTable 2 are set forth below.

PROTEIN CONSTRUCT SEQUENCES: MC-001(DNA)-SEQ ID NO: 52ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGCAAGCCATCATCATCACCACCACTAAMC-001 (protein)-(M)(UspA2 amino acids 30-540)(ASHHHHHH) SEQ ID NO: 53MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKASHHHHHH MC-002 (DNA)-SEQ ID NO: 54ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAATAAMC-002 (Protein)-(M)(UspA2 amino acids 30-540) SEQ ID NO: 55MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAY NELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTAL DTKSEQ ID NO: 56ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGAATGGCAGCACAAGCAGCACACTAA SEQ ID NO: 57MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAH MC-003 (DNA)-SEQ ID NO: 87ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAACACTAAMC-003 (Protein)-(M)(UspA2 amino acids 30-540)(H)-SEQ ID NO: 88MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKH MC-004 (DNA)-SEQ ID NO: 58ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAACATCATTAAMC-004 (Protein)-(M)(UspA2 amino acids 30-540)(HH) SEQ ID NO: 59MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKHH MC-005 (DNA)-SEQ ID NO: 60ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCGCAAGCCATCATCATCACCACCACTAAMC-005 (Protein)-(M)(UspA2 amino acids 30-519)(ASHHHHHH) SEQ ID NO: 61MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSASHHHHHHMC-006 (DNA)-SEQ ID NO: 62ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCTAAMC-006 (Protein)-(M)(UspA2 amino acids 30-519) SEQ ID NO: 63MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSMC-007 (DNA)-SEQ ID NO: 64ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCAGCAAGCCATCATCATCACCACCACTAAMC-007 (Protein)-(M)(UspA2 amino acids 30-564)(ASHHHHHH) SEQ ID NO: 65MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAASHHHHHH MC-008 (DNA)-SEQ ID NO: 66ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCACACCACTAAMC-008 (Protein)-(M)(UspA2 30-564)(HH) SEQ ID NO: 67MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH MC-009 (DNA)-SEQ ID NO: 68ATGGCGAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCACACCACTAAMC-009 (Protein)-(M)(UspA2 31-564)(HH) SEQ ID NO: 69MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH MC-010 (DNA)-SEQ ID NO: 70ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCATAAMC-010 (Protein)-(M)(UspA2 amino acids 30-564) SEQ ID NO: 71MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA MC-011 (DNA)-SEQ ID NO: 72ATGGCGAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGCAAGCCATCATCATCACCACCACTAAMC-011 (Protein)-(M)(UspA2 amino acids 31-540)(ASHHHHHH) SEQ ID NO: 73MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKASHHHHHH

Vector Construction and Transformation

DNA Sequence for UspA2 from strain ATCC 25238-SEQ ID NO: 74.ATGAAAACCATGAAACTTCTCCCTCTAAAAATCGCTGTAACCAGTGCCATGATTATTGGCTTGGGTGCGGCATCTACTGCGAATGCGCAGGCTAAAAATGATATAACTTTAGAGGATTTACCATATTTAATAAAAAAGATTGACCAAAATGAATTGGAAGCAGATATCGGAGATATTACTGCTCTTGAAAAGTATCTAGCACTTAGCCAGTATGGCAATATTTTAGCTCTAGAAGAGCTCAACAAGGCTCTAGAAGAGCTCGACGAGGATGTTGGATGGAATCAGAATGATATTGCAAACTTGGAAGATGATGTTGAAACGCTCACCAAAAATCAAAATGCTTTGGCTGAACAAGGTGAGGCAATTAAAGAAGATCTTCAAGGGCTTGCAGATTTTGTAGAAGGGCAAGAGGGTAAAATTCTACAAAATGAAACTTCAATTAAAAAAAATACTCAGAGAAACCTTGTCAATGGGTTTGAGATTGAGAAAAATAAAGATGCTATTGCTAAAAACAATGAGTCTATCGAAGATCTTTATGATTTTGGTCATGAGGTTGCAGAAAGTATAGGCGAGATACATGCTCATAATGAAGCGCAAAATGAAACTCTTAAAGGCTTGATAACAAACAGTATTGAGAATACTAATAATATTACCAAAAACAAAGCTGACATCCAAGCACTTGAAAACAATGTCGTAGAAGAACTATTCAATCTAAGCGGTCGCCTAATTGATCAAAAAGCAGATATTGATAATAACATCAACAATATCTATGAGCTGGCACAACAGCAAGATCAGCATAGCTCTGATATCAAAACACTTAAAAAAAATGTCGAAGAAGGTTTGTTGGAGCTAAGCGGTCACCTAATTGATCAAAAAACAGATATTGCTCAAAACCAAGCTAACATCCAAGATCTGGCCACTTACAACGAGCTACAAGACCAGTATGCTCAAAAGCAAACCGAAGCGATTGACGCTCTAAATAAAGCAAGCTCTGAGAATACACAAAACATCGAAGATCTGGCCGCTTACAACGAGCTACAAGATGCCTATGCCAAACAGCAAACCGAAGCAATTGACGCTCTAAATAAAGCAAGCTCTGAGAATACACAAAACATCGAAGATCTGGCCGCTTACAACGAGCTACAAGATGCCTATGCCAAACAGCAAACCGAAGCCATTGACGCTCTAAATAAAGCAAGCTCTGAGAATACACAAAACATTGCTAAAAACCAAGCGGATATTGCTAATAACATCAACAATATCTATGAGCTGGCACAACAGCAAGATCAGCATAGCTCTGATATCAAAACCTTGGCAAAAGCAAGTGCTGCCAATACTGATCGTATTGCTAAAAACAAAGCCGATGCTGATGCAAGTTTTGAAACGCTCACCAAAAATCAAAATACTTTGATTGAAAAAGATAAAGAGCATGACAAATTAATTACTGCAAACAAAACTGCGATTGATGCCAATAAAGCATCTGCGGATACCAAGTTTGCAGCGACAGCAGACGCCATTACCAAAAATGGAAATGCTATCACTAAAAACGCAAAATCTATCACTGATTTGGGCACTAAAGTGGATGGTTTTGACAGTCGTGTAACTGCATTAGACACCAAAGTCAATGCCTTTGATGGTCGTATCACAGCTTTAGACAGTAAAGTTGAAAACGGTATGGCTGCCCAAGCTGCCCTAAGTGGTCTATTCCAGCCTTATAGCGTTGGTAAGTTTAATGCGACCGCTGCACTTGGTGGCTATGGCTCAAAATCTGCGGTTGCTATCGGTGCTGGCTATCGTGTGAATCCAAATCTGGCGTTTAAAGCTGGTGCGGCGATTAATACCAGTGGTAATAAAAAAGGCTCTTATAACATCGGTGTGAATTACGAGTTCTAA

Protein Sequence for UspA2 from strain ATCC 25238.—SEQ ID NO. 1 asdescribed above.

Vector Construction

To generate the construct MC-001, DNA fragment coding for an UspA2 genefragment (amino acids 30 to 540 from strain ATCC 25238) including theNdeI/XhoI restriction sites to facilitate the cloning (the startingmethionine is encoded by NdeI site) and the DNA sequence correspondingto the AS (alanine serine) amino acids linker and 6×his amino acids wascodon-optimized (non-native) and synthesized by GENEART (a US registeredtrademark). Codon-optimized means that the nucleotide sequence waschanged from the native sequence without changing the amino acidsequence in order to better fit with the codon usage in Escherichia colifor optimal expression. The UspA2 fragment was cloned according tostandard methods into the pET-26b expression vector using the NdeI/XhoIrestriction sites.

To generate MC-002, MC-003, and MC-004 constructs, a polymerase chainreaction was performed to amplify the UspA2 gene fragment (amino acids30-540 from strain ATCC 25238) using the MC-001 construct as a template,the primer UspA2Nde opt (which contains the methionine start codon), andthe primer UspA2opt delta His, A2opt 1His delta AS, and A2opt 2His deltaAS, respectively. The UspA2 fragment was cloned according to standardmethods into the pET-26b expression vector using the NdeI/XhoIrestriction sites.

To generate the construct MC-005, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 30-519 fromstrain ATCC 25238) using the MC-001 vector as a template with theprimers UspA2Nde opt (which contains the methionine start codon) and Rdelta hairpin A2opt His. DNA sequence corresponding to NdeI restrictionsite was incorporated into the 5′ primer and XhoI restriction site wasincorporated into the 3′ primer. In addition, DNA sequence correspondingto the AS amino acids linker and 6×his amino acids was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN (a US registered trademark)).

To generate the construct MC-006, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 30-519 fromstrain ATCC 25238) using the MC-005 construct as a template with theprimers UspA2Nde opt (which contains the methionine start codon) anddelta His delta hélice. DNA sequence corresponding to NdeI restrictionsite was incorporated into the 5′ primer and XhoI restriction site wasincorporated into the 3′ primer. The generated PCR product was theninserted into the pET-26b(+) cloning vector (NOVAGEN (a US registeredtrademark)).

To generate the construct MC-007, DNA fragment coding for an UspA2 genefragment (amino acids 30 to 564 from strain ATCC 25238) including theNdeI/XhoI restriction sites to facilitate the cloning (startingmethionine is encoded by NdeI site) and the DNA sequence correspondingto the AS amino acids linker and 6×his amino acids was codon-optimizedand synthesized by GENEART (a US registered trademark) (plasmid:1026399). The UspA2 fragment was cloned according to standard methodsinto the pET-26b expression vector using the NdeI/XhoI restrictionsites.

To generate the construct MC-008, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 30-564 fromstrain ATCC 25238) using the MC-007 construct as a template with theprimers UspA2Nde opt (which contains the methionine start codon) and2His hélice deltaAS. DNA sequence corresponding to NdeI restriction sitewas incorporated into the 5′ primer and XhoI restriction site wasincorporated into the 3′ primer. The generated PCR product was theninserted into the pET-26b(+) cloning vector (NOVAGEN (a US registeredtrademark)).

To generate the construct MC-009, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 31-564 fromstrain ATCC 25238) using the 1026399 plasmid as the template and theprimers N-term cyto Abis (which contains the methionine start codon) and2His hélice deltaAS. DNA sequence corresponding to NdeI restriction sitewas incorporated into the 5′ primer including the glutamine deletion andXhoI restriction site was incorporated into the 3′ primer including twohistidine residues. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN (a US registered trademark)). DNAsequencing of the final construct was performed to confirm the correctsequence.

To generate the construct MC-010, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 30-564 fromstrain ATCC 25238) using the MC-007 construct as a template with theprimers UspA2 Nde opt (which contains the methionine start codon) andcyto hélice dHis dAS. DNA sequence corresponding to NdeI restrictionsite was incorporated into the 5′ primer and XhoI restriction site wasincorporated into the 3′ primer. The generated PCR product was theninserted into the pET-26b(+) cloning vector (NOVAGEN (a US registeredtrademark)).

To generate the construct MC-011, a polymerase chain reaction wasperformed to amplify the UspA2 gene fragment (amino acids 31-540 fromstrain ATCC 25238) using the MC-001 construct as a template with theprimers N-term cyto Abis (which contains the methionine start codon) andN-term reverse. DNA sequence corresponding to NdeI restriction site wasincorporated into the 5′ primer and XhoI restriction site wasincorporated into the 3′ primer. The generated PCR product was theninserted into the pET-26b(+) cloning vector (NOVAGEN (a US registeredtrademark)).

A detailed list of PCR primer sequences used for amplifications isillustrated in Table 3. The polymerase chain reaction was performedusing Expand High Fidelity PCR System kit (Roche) according tomanufacturer's recommendations. Ligation was performed using Rapid DNALigation Kit (Roche) according to manufacturer's recommendations.

TABLE 3 PCR primer sequences used for UspA2 amplifications DNA SequencePrimer ID 5′-3′ UspA2 Nde optGAATTCTTAATTAACATATGCAGGCCAAAAATGATATTACCCTG (SEQ ID NO: 75)UspA2opt deltaGGCGCGCCTCGAGTTATTATTTGGTATCCAGTGCGGTCACACG (SEQ ID NO: 76) HisUspA2opt 1HisGGCGCGCCTCGAGTTAGTGTTTGGTATCCAGTGCGGTCACACG (SEQ ID NO: 77) delta ASUspA2opt 2His GGCGCGCCTCGAGTTAGTGGTGTTTGGTATCCAGTGCGGTCACACG (SEQ IDdelta AS NO: 78) R delta hairpinGGCGCGCCTCGAGTTAGTGGTGGTGATGATGATGGCTTGCGCTTTTGGCATTTTTG A2opt HisGTGATGGCAT (SEQ ID NO: 79) Delta His deltaCCGCTCGAGCTAGCTTTTGGCATTTTTGGTGATGGC (SEQ ID NO: 80) héliceN term cytoAbisGGAATTCCATATGGCGAAAAATGATATTACCCTGGAAGATCTG (SEQ ID NO: 81)2His hélice delta GGCGCGCCTCGAGTTAGTGGTGTGCTGCCTGTGCTGCCATACCATT (SEQ IDAS NO: 82) Cyto hence dHisGGCGCGCCTCGAGTTATGCTGCCTGTGCTGCCATACCATT (SEQ ID NO: 83) dASN term reverse CAGTTCATTATAGGTGGCCAGATCCTG (SEQ ID NO: 84)

Transformation

Escherichia coli (E. coli) BLR (DE3), modified BLR (DE3) or B834(DE3)cells were transformed with plasmid DNA according to standard methodswith CaCl₂-treated cells (Hanahan D. «Plasmid transformation bySimanis.» In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985):p. 109-135). Briefly, BLR (DE3) competent cells were gently thawed onice. Approximately 4 μl of plasmid (10-100 ng) were mixed using 50-100μl competent cells. Thereafter, this formulation was incubated on icefor 5 min. To perform the transformation reaction, the formulation washeat pulsed at 42° C. for 30 seconds then incubated on ice for 2minutes. Approximately 0.5 ml of SOC medium (Super Optimal broth withCatabolite repression) was added to the transformed cells and the cellculture was incubated at 37° C. for one hour before plating onLuria-Bertani (LB) agar with 50 μg/ml kanamycin. Around 150 μl oftransformed cell culture was plated and incubated overnight at 37° C.

BLR (DE3): BLR is a recA⁻ derivative of BL21 (F- ompT hsdSB(rB- mB-) galdcm (DE3). This E. coli strain used for expression of recombinantproteins improves plasmid monomer yields and may help stabilize targetplasmids containing repetitive sequences or whose products may cause theloss of the DE3 prophage (Studier, F. W. (1991) J. Mol. Biol. 219:37-44). The detailed genotype of E. coli BLR (DE3) has been published byNOVAGEN (a US registered trademark). (F- ompT hsdSB (rB- mB-) gal dcmΔ(srl-recA)306::Tn10 (TetR) (DE3).

B834 (DE3) is the parental strain for BL21. These hosts are methionineauxotrophs and allow high specific activity labeling of target proteinswith 35S-methionine and selenomethionine for crystallography. Thedetailed genotype of E. coli B834 (DE3) has been published by NOVAGEN (aUS registered trademark): F⁻ ompT hsdS_(B)(r_(B)- m_(B)-) gal dcm met(DE3).

Modified BLR (DE3): In order to prevent (phospho)gluconoylation, Pglgene was inserted in the biotin locus located in the BLR (DE3) genome.In addition, to prevent the Ile-Val substitutions, the C219Y mutation inthe threonine deaminase gene was corrected.

-   -   Genotype: (F- ompT hsdSB (rB- mB-) gal dcm Δ(srl-recA)306::Tn10        (TetR); Δ(bioA-bioD)::Pgl; TD+(C21919 (DE3).

Example 2: Protein Expression Using Shake Flask

Escherichia coli strains transformed with recombinant plasmid were usedto inoculate 100 ml of LB broth (Becton, Dickinson and Company)±1%(weight/volume, w/v) glucose (Laboratoire MAT, catalogue number:GR-0101) and 50 μg/ml kanamycin (Sigma). This preculture was generallygrown overnight at 37° C. Twelve ml of the preculture is used toinoculate 500 ml LB broth+50 μg/ml kanamycine. Cultures were incubatedat 37° C. with agitation of 150 RPM to reach an O.D._(600nm) of ˜0.6.

At an O.D._(600nm) ˜0.6, the BLR (DE3) cultures were induced for theexpression of the recombinant protein by addition of 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG; EMD Chemicals Inc., catalogue number:5815) and incubated overnight at 23° C. with agitation of 150 RPM. Afterthe induction period, the cultures were centrifuged at 6370 g for 20minutes and the pellets from 350 ml culture were frozen at −20° C.separately.

Example 3: Protein Purification Using Phosphate Buffer (MC-001 Constructand MC-011 Construct)

Each bacterial pellet obtained after induction in shake flask wasresuspended in 30 ml 20 mM potassium phosphate buffer (pH 8.0)containing 10 mM NaCl and Roche COMPLETE (a US registered trademark)Protease Inhibitor Cocktail (1 tablet/50 ml buffer). Cell lysis isperformed by 3× French Press extractions (20 000 psi) and clarificationis performed by 30 minutes centrifugation at 23700 g. Supernatant isharvested and filtrated on 0.22 μm.

6×His tagged-proteins were purified on immobilized metal affinitychromatography (IMAC) using XK16 column and 20 ml NiNTA resin (Qiagen)previously equilibrated with 20 mM potassium phosphate buffer (pH 8.0)containing 10 mM NaCl or PBS buffer pH 8.0 containing 500 mM arginine.The soluble components were loaded on at up to 4 ml/min (producing a“flow through fraction”). After loading on the column, the column waswashed with 60 ml of 20 mM potassium phosphate buffer (pH 8.0)containing 10 mM NaCl at a rate of 4 ml/min producing a “wash fraction#1. A second wash using same buffer+10 mM imidazole was performed,producing a “wash fraction #2. Elution was performed using same buffercontaining 200 or/and 500 mM imidazole.

Samples from elution fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Samples containing theprotein were dialyzed against 5 liters of 20 mM potassium phosphatebuffer (pH 8.0) containing 10 mM NaCl. Protein concentration wasdetermined using Lowry method.

Example 4: Protein Purification Using Arginine Containing Buffer(MC-001, MC-005 and MC-007)

Each bacterial pellet obtained after induction in shake flask (Example3) or fermenter (Example 5) was resuspended in 30 ml PBS buffer+500 mMarginine pH8.0 and Roche COMPLETE (a US registered trademark) ProteaseInhibitor Cocktail (1 tablet/50 ml buffer). Alternatively, fermentationcell paste (≈7 g) was resuspended in 90 ml PBS buffer containing 500 mMarginine pH8.0 and Roche COMPLETE (a US registered trademark) ProteaseInhibitor Cocktail (1 tablet/50 ml buffer).

Cell lysis was performed by 2 or 3× French Press extractions (20 000psi) and clarification was performed by 30 minutes centrifugation at 23700 g 4° C. Supernatant was harvested and filtrated on 0.22 μm. 6×Histagged-proteins were purified on immobilized metal affinitychromatography (IMAC) using XK16 column and 80 ml NiNTA resin (Qiagen)previously equilibrated with PBS buffer+500 mM arginine pH 8.0. Thesoluble components were loaded on at up to 4 ml/min (producing a “flowthrough fraction”). After loading on the column, the column was washedwith the same buffer, then with 20 mM potassium phosphate buffer (pH8.0) containing 10 mM NaCl at a rate of 4-6 ml/min producing a “washfraction #1.” A second wash using same buffer+10 mM imidazole wasperformed, producing a “wash fraction #2.” Elution was performed usingsame buffer+200 mM imidazole or 500 mM imidazole. In further elutionvials, 5 mM EDTA final concentration was added.

Samples from elution fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Samples containing theproteins were dialyzed against 5 liters of 20 mM potassium phosphatebuffer (pH 8.0) containing 10 mM NaCl and 5 mM EDTA. Proteinconcentration was determined using Lowry method.

This protocol may be used with other 6×His tagged-proteins.

Example 5: Fermentation

The following fermentation procedure may be used:

Working seeds are frozen aliquots of flask-grown Escherichia coliBLR(DE3) or BLR(DE3)-derived strains transformed with a pET26bderivative containing a sequence coding for a specific antigen candidaterecombinant protein construct.

A working seed (WS) is removed from frozen storage, thawed and used toinoculate an Erlenmeyer flask containing pre-culture media. Manipulationof the seed and flask culture are performed aseptically under a LaminarAir Flow (LAF) Hood or Biological Safety Cabinet (BSC). The pre-cultureflask is incubated typically between 30° C.-37° C. under 200 RPMagitation speed for the time needed to reach an Optical Density at 650nm (OD_(650nm)) between 1.0 and 3.0, typically between 4-6 hrs.

A 20 L fermentor is prepared by Clean-In-Place followed by an automatedsteam sterilisation sequence. Starting medium is transferred asepticallyinto the fermentor. A bottle filled with NH₄OH 25% is asepticallyconnected to the fermentor for automatic pH control. The initial pH ofthe starting medium is adjusted to target pH by addition of NH₄OHsolution. Irradiated antifoam is added using a syringe through a septumin the head-plate. A bottle filled with Feed medium is asepticallyconnected to the fermentor. Feed addition is controlled by either apO2-cascade (control dissolved oxygen) to or a pre-programmedfeed-curve. Agitation is controlled either by a pO2-cascade or apre-programmed agitation-curve.

Initial fermentor parameters are typically as follows:

-   -   Temperature: 28° C.-32° C.    -   Pressure: 0.5 barg (7 psi)    -   Air flow rate: 2 VVM (Vessel Volumes per Minute)    -   pH: Regulated at 6.8 by addition of NH₄OH 25%.

An aliquot of this pre-culture (typically between 5 ml-50 ml) is used toinoculate the starting fermentor media by syringe addition through aseptum on the fermentor head-plate. The phases of Fermentation Cultureare:

-   -   Batch Phase: Biomass is accumulated using carbon source in        starting media.    -   Fed-batch Phase: Feed media is introduced either according to        pO2-cascade control or pre-programmed feed curve. Biomass        accumulation continues on carbon source in feed media.    -   Induction Phase: Expression of the recombinant protein antigen        is induced by addition of IPTG solution to the culture in the        fermentor.

At harvest, the culture is collected typically in 1 L centrifugationbottles and centrifuged to separate the solid pellet (cell-paste)fraction from the liquid supernatant fraction. The supernatant isdiscarded, and the wet cell weight (solid pellet) is recorded and thecell-paste bags stored at −20° C.

The following procedure may also be used:

Escherichia coli Standard Pre-Culture

Each standard pre-culture were prepared using a frozen seed culture ofEscherichia coli strains. These strains are BLR(DE3) strains transformedwith a pET26b derivative containing a sequence coding for the specificconstruct to be evaluated.

The seed culture was thawed to room temperature and 400 μl were used toinoculate a 2 litre Erlenmeyer flask containing 400 ml of preculturemedium (adapted from Zabriskie et al. (J. Ind. Microbiol. 2:87-95(1987)).

The inoculated flask was then incubated at 37° C. (±1° C.) and 200 rpm.The pre-culture was stopped after 6 h of incubation. At this step theoptical density at 650 nm (OD_(650nm)) is about 2. The pre-culture wasused to inoculate medium in a fermenter as soon as the culture wasstopped.

20L Scale Fedbatch Fermentation

Method

A 20 litre fermenter (Biolafitte) was used. Nine litres of batch phasemedium were aseptically transferred into the fermenter. The pH of themedium was readjusted to 6.8 with base addition. 1 ml of undilutedirradiated antifoam (SAG 471) was also added to the fermenter. Thetemperature (28° C.), head pressure (0.5 bar), aeration rate (20 litressparged air per minute) and initial agitation speed (300 rpm) were thenset prior to inoculation. The level of dissolved oxygen in theseconditions was 100%. The head pressure and aeration rate were maintainedat a constant level during the fermentation.

Inoculation was achieved by the addition of an equivalent 10 ml OD650nm=2 of pre-culture (prepared as described above, in Example 2)following the next formula:

${{Preculture}\mspace{14mu}{Volume}\mspace{14mu}({ml})} = \frac{20}{{Preculture}\mspace{14mu}{Final}\mspace{14mu}{OD}\; 650\mspace{14mu}{nm}}$

During batch phase (0-15 h), the temperature was maintained at 28° C.The level of dissolved oxygen was set at 20%. The level of dissolvedoxygen (DO) was regulated by increasing stirring when the DO fell below20%. Glucose exhaustion resulted in an increase in DO and a concomitantdecrease in stirring.

When glucose is exhausted, the feeding rate is started based on a pHsignal that increases above 7.0. From this point forward, the feedingrate was controlled by oxygen demand, increasing the flow rate whendissolved oxygen tends to drop below the 20% set point. At this step theagitation speed is maintained at 900 rpm.

During the fed-batch phase (before induction), the pH was maintained at6.8 by addition of base, the temperature was regulated at 30° C.

Two strategies were applied to produce the protein:

The “High Cell Density Induction” (HCDI) is applied when culture isinduced with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) at anoptical density of 80±5, typically reached after 40 h of culture. Thetemperature was maintained at 28° C. and feeding rate still controlledby oxygen demand with a constant agitation speed at 900 rpm.

The “Low Cell Density Induction” (LCDI) process means an induction at anoptical density of 40±5 usually reached after 24 h of culture. Thetemperature was decreased to 30° C. and the constant feeding rate of 0.5ml/min is applied. Then 1 mM IPTG is added to the culture. At this step,the DO level was maintained at 20% by controlling the stirring rate.

At the end of the induction phase (72 h), cell paste was collected bycentrifugation (6,500×g, 4° C. for 1 h), and stored at −20° C.

FIGS. 1 and 2 illustrate a typical fermentation profile with the HCDIand the LCD processes and the parameters monitored during 20 L-scalefed-batch fermentation.

Table 4 sets forth the constructs evaluated in fermenter and UspA2 yieldobtained for each one.

TABLE 4 UspA2 Construct His Process Yield ID Name Helix tag used (g/l)MC-008 UspA2 + Helix + 2 His Full 2 res HCDI 2.21 MC-007 UspA2 + Helix +6 His Full 6 res LCDI 2.60 MC-010 UspA2 + Helix Full No HCDI 0.22 MC-005UspA2 ΔHelix + 6 His No 6 res LCDI 1.92 MC-006 UspA2 ΔHelix No No LCDI1.14 MC-004 UspA2 + ½ Helix + 2 His ½ 2 res LCDI 0.92 MC-001 UspA2 + ½Helix + 6 His ½ 6 res HCDI 3.68 MC-002 UspA2 + ½ Helix ½ No HCDI 0.49His = histidine

FIG. 3 depicts in graphical form the UspA2 yield in Table 4 from theconstructs evaluated in fermenter.

In this figure, UspA2 yield is affected by histidine residues present inthe construct. (p<0.05, one way, three levels, Type II ANOVA). Apositive correlation between the number of histidine residues and UspA2fermentation yield was observed, with a yield increase higher than 400%between 0 and 6 residues in fed-batch fermentations.

It was also observed that one histidine residue added to half-helixpattern (MC-003 construct) produced a UspA2 yield of about 1 g/l ofprotein.

Example 6: Protein Characterization

Analytical Ultracentrifugation

Analytical ultracentrifugation is used to determine the homogeneity andsize distribution in solution of the different species within a proteinsample by measuring the rate at which molecules move in response to acentrifugal force. This is based on the calculation of the coefficientsof sedimentation of the different species that are obtained bysedimentation velocity experiment, which depend on their molecular shapeand mass.

The following protein samples were spun in a Beckman-Coulter ProteomeLabXL-1 analytical ultracentrifuge at 28 000RPM after the AN-60Ti rotor hadbeen equilibrated to 15° C.

a. MC-005 lot BMP53, 675 μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0

b. MC-001 lot BMP13, 545 μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0

c. MC-001 lot BMP14, 545 μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0

d. MC-001 lot BMP54, 445 μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0

e. MC-007 lot BMP70, 510 μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0

For data collection, from 133 to 325 scans were recorded at 280 nm every5 minutes.

Data analysis was performed using the program SEDFIT (available throughthe National Institutes for Health) for determination of the C(S)distribution. The C(S) distribution is a representation of the relativeintensity of the different components in a mixture of macromoleculesseparated by their coefficient of sedimentation, which is a function ofmolecule size and conformation. Determination of the partial specificvolume of the proteins at 15° C. was performed with the SEDNTERPsoftware from their amino acid sequence. SEDNTERP (SEDNTERP isdistributed and supported through the Biomolecular InteractionTechnologies Center at the University of New Hampshire) was also used todetermine the viscosity and the density of the buffer at 15° C.

Determination of the relative abundance of all species has beenperformed by considering the total area under the curve of the overalldistribution as 100% of the sample and by calculating the percentage ofthis total area represented by the contribution of every species. C(S)distribution plot (concentration vs sedimentation coefficient) has beenused for that calculation, considering that it's a better representationof the raw data than the C(M) distribution (concentration vs molecularweight).

Analytical ultracentrifugation of the different purified constructsallowed observation that UspA2 Δhelix, UspA2½ helix and UspA2 full helixwith C-terminal his tag are present mainly as trimers in solution when500 mM L-arginine is added during cell lysis prior to purification(FIGS. 4, 5, 7 and 8).

A heterogeneous size distribution has been observed for UspA2½ helixwhen no L-arginine was added during cell lysis. Two major populationsare observed. It was not possible to confirm the molecular weight of thespecies detected by AUC (analytical ultracentrifugation) with thisprotein preparation, since the frictional ratio which is essential formolecular weight estimation needs to be calculated from an homogeneoussample. However, based on sedimentation coefficients, none of theobserved species seem to correspond to the trimer observed in othersamples.

FIG. 4 illustrates the molecular weight distribution of purified MC-005determined by sedimentation velocity analytical ultracentrifugation. Themajority of protein is found as a trimer, with a small proportion of ahigher molecular weight oligomer that may correspond to dimer of trimer.

FIG. 5 illustrates the molecular weight distribution of purified MC-001determined by sedimentation velocity analytical ultracentrifugation.Majority of protein is found as a trimer.

FIG. 6 illustrates the molecular weight distribution of purified MC-001determined by sedimentation velocity analytical ultracentrifugation. Thesample presents multiple species and is highly polydisperse. Thesedimentation coefficient of the major species detected doesn'tcorrespond to the one of the trimers normally detected in the otherlots.

FIG. 7 illustrates the molecular weight distribution of purified MC-001determined by sedimentation velocity analytical ultracentrifugation. Themajority of protein is found as a trimer.

FIG. 8 illustrates the molecular weight distribution of purified MC-007determined by sedimentation velocity analytical ultracentrifugation. Themajority of protein is found as a trimer.

Circular Dichroism/Secondary Structure

Circular dichroism (CD) is used to determine the secondary structurecomposition of a protein by measuring the difference in the absorptionof left-handed polarized light versus right-handed polarized light whichis due to structural asymmetry. The shape and the magnitude of the CDspectra in the far-UV region (190-250 nm) are different whether aprotein exhibits a beta-sheet, alpha-helix or random coil structure. Therelative aboundance of each secondary structure type in a given proteinsample can be calculated by comparison to reference spectra.

Far UV spectra are measured using an optical path of 0.01 cm from 178 to250 nm, with a 1 nm resolution and bandwidth on a Jasco J-720spectropolarimeter. Temperature of the cell is maintained at differenttemperatures by a Peltier thermostated RTE-111 cell block. A nitrogenflow of 10 L/min is maintained during the measurements.

Concentration of the following protein constructs was adjusted to 400μg/ml in 20 mM NaPO₄, 10 mM NaCl, pH8.0 buffer.

a. MC-005 lot BMP53, in 20 mM NaPO₄, 10 mM NaCl, pH8.0

b. MC-001 lot BMP13, in 20 mM NaPO₄, 10 mM NaCl, pH8.0

c. MC-001 lot BMP14, in 20 mM NaPO₄, 10 mM NaCl, pH8.0

d. MC-001 lot BMP54, in 20 mM NaPO₄, 10 mM NaCl, pH8.0

e. MC-007 lot BMP70, in 20 mM NaPO₄, 10 mM NaCl, pH8.0

Calculations of secondary structures have been done using the followingalgorithms:

-   -   Selcon 3 (Sreerama and Woody, Anal. Biochem. (1993), 209, 32;        Sreerama and Woody, Biochemistry, 33, 10022-25 (1994); Sreerama        et al. Protein Science, 8, 370-380 (1999); Johnson W. C. Jr.,        Proteins: Str. Func. Genet. 35, 307-312 (1999))    -   CDSSTR (Johnson W. C. Proteins: Struc. Func. Genet. 35,        307-312 (1999) modified by Sreerama. N. (Anal. Biochem., 287,        252 (2000)).

Displayed results are an average of the percentage calculated with bothalgorithms and are subjected to a 5% error margin.

Results of the secondary structure calculations for fermentor expressedproteins are displayed in Table 5, considering a 5% error margin.

TABLE 5 Secondary structure calculations at 22° C. Protein Helix Betarandom MC-005 BMP53 40.8 26.4 34.1 MC-007 BMP70 58.2 18.2 24.7 MC-001BMP54 53.7 14.2 34.4

Calculations are compatible with the shape and visual analysis of thespectra, where helical content increases with the intensity of minima at208 and 220 nm. Proteins are composed of a high proportion of helicalstructures, with presence of beta structures.

Superposition of the spectra on FIG. 9 shows no significant differencein the shape between constructs. Spectra of MC-005 helix shows a lowerintensity which could account for a lower alpha structure that iscoherent with the absence of C-terminal helix.

FIG. 9 illustrates the far-UV circular dichroism spectra of UspA2constructs MC-001, MC-005 and MC007 giving an indication of proteinsecondary structures. Superposition of spectra clearly shows thatconstructs containing half and full C-terminal helix have no detectabledifference in their secondary structures, while the construct withouthelix generates a spectra with a difference in intensity that couldaccount for a different secondary structure content.

Thermal Unfolding

Measurement of far-UV CD spectra at different temperatures duringthermal unfolding suggested that MC-005 is less thermally stable thanMC-007. The spectra observed at 33° C. for MC-005 is similar to thetypical spectra of an unfolded protein. For the MC-007 construct, evenif partial loss of secondary structures is observed at 33° C., thecomplete unfolding seems to occur between 35° C. and 37° C. This may bean indication of higher thermal stability of the full helix containingconstruct MC-007.

FIG. 10 illustrates secondary structures monitoring by circulardichroism during thermal unfolding of MC-005 (UspA2Δhelix+6His). Visualanalysis of the spectra clearly shows that the protein loses most of itssecondary structures at 33° C.

FIG. 11 illustrates secondary structures monitoring by circulardichroism during thermal unfolding of MC-007 (UspA2+helix+6His). Visualanalysis of the spectra shows that loss of secondary structure is slowercompared to the construct without helix. Structural changes aredetectable upon heating to 33° C., but complete unfolding seems to occurbetween 35° C. and 37° C.

Differential Scanning Calorimetry (DSC) Thermal Unfolding

Thermal transitions of different UspA2 constructs were compared in orderto evaluate the effect of C-terminal helix modifications on thermalstability of the proteins.

Analysis was done on VP-DSC from MicroCal (part of GE Healthcare). Thebuffer 20 mM NaPO₄, 10 mM NaCl, 5 mM EDTA, pH8 was used as reference andsubtracted from the scans. Proteins were equilibrated at initialtemperature for 15 minutes before temperature ramping DSC scans werethen conducted from 10° C. to 60° C. at a heating rate 90° C./hr.

Two transitions were detected in MC-001 and MC-007 constructs and onlyone in MC-005. Values of the transitions (or Tm) of the differentconstructs can be found on Table 6.

While the lower Tm of all three proteins is around 32° C., the maindifference is the value of the second Tm. The construct containing afull helix (MC-007) has a higher Tm at 37.5° C. compared to 34.5° C. forthe half helix (MC-001).

It has been demonstrated that for MC-001 and MC-007, the first Tm around32° C. is reversible, while the higher Tm is irreversible. For MC-005,the only Tm detected is irreversible.

This may be an indication of a higher thermal stability of full helixcontaining construct MC-007.

TABLE 6 Melting points of UspA2 constructs measured by DSC Constructs[mg/mL] Tm₁ (° C.) Tm₂ (° C.) MC-005 0.400 31.74 — lotBMP53 MC-001 0.40032.02 34.51 lotBMP54 MC-007 0.400 32.19 37.50 lotBMP70

Mass Spectrometry

UspA2 protein samples were prepared by protein precipitation withCHCl₃/MeOH/H₂O system. The protein pellet was centrifuged at the bottomof the eppendorf tube before being gently dried under nitrogen. Thedried pellet was then dissolved in 2 μl of pure formic acid before beingdiluted with 3 μl of ultrapure water and 5 μl of sinapinic acid.Sinapinic acid used as matrix for MALDI-TOF (Matrix-Assisted LaserDesorption/lonisation followed by Time-Of-Flight spectrometry analyser)analysis is prepared in 50% CH₃CN/50% H₂O supplemented by TFA 0.1% finalconcentration.

1 μl of the sample+matrix mixture was spotted onto a Bruker 384 groundstainless steel MALDI target and let to dry for crystallization at roomtemperature and atmospheric pressure (dried droplet method).

UspA2 mass spectrometry analysis was performed on a Bruker Ultraflex 2MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) inpositive ionization and linear mode. Protein samples, co-crystallized insinapinic acid matrix, were irradiated by a smartbeam laser. Massmeasurement of intact UspA2 protein were done over 10.000-100.000 Damass range with an acceleration voltage of 25 kVolts. Laser attenuationwas fine-tuned in order to get the best protein signal as possible andto avoid any fragmentation as well as background over-ionizationphenomena. Calibration of the mass spectrometer was performed in closeexternal method with homologous matrix and using the commercial BrukerProtein Calibration mixture 2, by accurate measures on the followingcalibrators: [M+2H]²⁺ (mass measured by MS detector following additionof two H+ ions to the protein during ionisation) species of protein A atm/z 22307 Da, [M+H]⁺ species of trypsinogen at m/z 23982 Da,[M+H]+species of protein A at m/z 44613 Da and [M+H]+species of bovinealbumin at m/z 66431 Da°. Each presented spectrum results from the sumof 500 individual shots.

The following samples were analyzed:

MC-001 construct with MQAK amino acids (SEQ ID NO: 85) in N-terminalproduced in shake flask, lot opt-01, MC-011 construct with MAK aminoacids in N-terminal produced in shake flask, lot BMP37.

In Table 7 and FIG. 12, MC-001 protein with MQAK amino acids (SEQ ID NO:85) at the N-terminal extremity has been shown to be at least partiallydemethionylated, as shown by the measured molecular mass of 57427 Da,compared to the expected mass of 57565 Da. The other peak of 57620 Damay represent the complete non-demethionylated protein, N-acetylatedprotein, or another modified protein population.

FIG. 12 illustrates the MALDI spectrum of MC-001 lot opt-01. The massobserved at 57427 Da may be coherent with the demethionylated protein,while the peak at 57620 Da could correspond to the complete protein.

As shown in Table 7 and FIG. 13, MC-011 protein with MAK amino acids atthe N-terminal extremity gave a major population in MALDI-MS that maycorrespond to the demethionylated protein, with a mass of 57265 Da,compared to 57437 Da for the expected mass based on complete amino acidsequence. The two other peaks at +186 Da and +366 Da are not close toany expected post-translational modifications, so they couldn't beidentified by this experiment.

TABLE 7 Molecular mass of two UspA2 constructs as measured by MALDI-MS.Both constructs have a main measured mass lower than the one expectedfrom amino acid sequence. The mass of the major population obtained withboth constructs may be coherent with a demethionylated protein.Theoretical Measured Protein mass (Da) mass (Da) Comment MC-001 57565.8−57427.9 Coherent with demethionylation lot opt-01 (57434.6) −57620.3Coherent with protein containing N-terminal methionine MC-011 lot57437.6 57265.2 Coherent with demethionylation BMP37 (57306.4)

N-Terminal Sequencing by Edman's Degradation

In order to evaluate if the optimisation of the N-terminal region(optimisation of the amino acid sequence next to the N-terminalmethionine) leads to demethionylation of the protein, N-terminalsequencing has been done on the MC-011 construct carrying MAK aminoacids on his N-terminal extremity.

The proteins were separated by SDS PAGE on a Novex 4%-20% polyacrylamidegel from Invitrogen, before transfer onto Problot PVDF (polyvinylidencediluoride) (Bio-Rad) membrane. The membrane was stained with amidoblack.The band of interest was then cut and analysis was carried out accordingto the manufacturer's protocol using an Applied Biosystems Procisesequencer system. Twelve cycles of Edman's degradation were performed.

The N-terminal amino acid sequence obtained is AKNDITLEDLP (SEQ IDNO:86), which corresponds to the N-terminal extremity of the proteinstarting at the amino acid number two after the initial methionine. Thisindicates that the mature protein is mainly demethionylated.

Example 7: UspA2 Construct MC-001: Bactericidal Activity

Bactericidal Assay

Moraxella catarrhalis was cultivated overnight on Petri dish at 37°C.+5% CO2. Bacteria were transferred in 12 ml HBSS-BSA (Hank's BufferedSalt Solution with Bovine Serum Album) 0.1% buffer in order to get anOD₆₂₀ of 0.650. Serum samples were heated for 45 min at 56° C. toinactivate the endogenous complement. Serial two-fold dilutions of serain SBA buffer (HBSS-BSA 0.1%) were added on a 96-well round bottommicrotitre plate (25 μl/well). Subsequently, 50 μl of SBA buffer wereadded in each well. Then 25 μl of Moraxella catarrhalis strains at 4 10⁴cfu/mL were added to the wells containing sera and incubated for 15 minat room temperature. Finally 25 μl of freshly thawed baby rabbitcomplement diluted ⅛ in HBSS-BSA 0.1% were added to reach a final volumeof 125 μl. Plates were incubated for 1 h at 37° C. with orbital shaking(210 rpm). The reaction was stopped by laying the microplate on ice forat least 5 min.

After homogenization, various dilutions of the suspension (a mixture ofbacteria, serum, complement and buffer, at a volume of 125 μl asdiscussed in the previous paragraph) were added onto chocolate agarplates and incubated for 24 hours at 37° C. with 5% CO₂ and Moraxellacatarrhalis colonies were counted.

Eight wells without serum sample were used as bacterial controls todetermine the number of Moraxella catarrhalis colonies per well. Themean number of CFU (colony forming unit) of the control wells wasdetermined and used for the calculation of the killing activity for eachserum sample. The bactericidal titers were expressed at the reciprocaldilution of serum inducing 50% of killing.

Anti-UspA2 antisera generated in mice, guinea pigs and rabbits againstMC-001 were tested in the bactericidal assay described here aboveagainst 20 different Moraxella catarrhalis strains isolated from varioustissues (blood, sputum, nose, middle ear fluids) in various countries(US, Finland, Netherlands, Norway, Sweden).

As shown below anti-UspA2 antibodies were able to induce across-bactericidal killing of Moraxella catarrhalis, whatever thepercentage of homology of the UspA2 expressed by the tested strain.Moreover bactericidal activity was also shown against strains which onlyexpress UspA1 or the chimeric protein UspA2H. As expected, no or onlyweak bactericidal antibody titres were measured against UspA1 and UspA2double knock-out mutants.

TABLE 8 Cross-bactericidal activity of anti-UspA2 MC-001 antibodiesgenerated in mouse, guinea pig and rabbit. 1 + 2 KO is a double knockout, UspA1 & UspA2. 1KO is a UspA1 knockout only. MEF (AOM) = Middle EarFluid (Acute Otitis Media). “/” in the Isolate source column = not awareof the isolate source. Identity %* versus the Anti-UspA2 antiserum UspAgene vaccine sequence bactericidal activity Strains Isolate sourcepresent ATCC25238 Mouse Guinea pig Rabbit ATCC 25238 / UspA1/UspA245.2/100  +++ ++ +++ 43617 Bronchitis UspA1 40.1 + + + American 2926 /UspA1 36.4 − +/− − 2933 / UspA1/UspA2 44.6/62.4 − ++ ++ 2912 /UspA1/UspA2 39.3/64.6 ++ ++ +++ 2908 / UspA1/UspA2 43.3/52.8 +++ +++ +++Finnish 307 MEF (AOM) UspA1/UspA2 47.6/70.1 − ++ +++ 353 MEF (AOM)UspA1/UspA2  45/61.8 + ++ ++ 358 MEF (AOM) UspA1/UspA2  47/61.5 +++ ++++++ 216 MEF (AOM) UspA1/UspA2 46.6/66.9 +++ +++ +++ Dutch N9 noseUspA1/UspA2H 41.1/70.1 ++ +++ +++ H2 sputum UspA1/UspA2  47/61.6 +++ ++++++  F10 sputum UspA1/UspA2 42.9/61.1 ++ +++ +++ Norvegian 1 Tracheotomy(Pneumonia) UspA1/UspA2 44.6/60.9 +++ +++ +++ 13 Tracheotomy (Pneumonia)UspA1/UspA2 47.8/55  ++ +++ +++ 20 Tracheotomy (Pneumonia) UspA1/UspA233.7/60.6 +++ ++ +++ 25 Tracheotomy (Pneumonia) UspA1/UspA2 47.8/55  +++++ +++ 27 Tracheotomy (Pneumonia) UspA1/UspA2  46/76.1 ++ +++ +++ 36Tracheotomy (Pneumonia) UspA1/UspA2 53.6/61.6 + ++ ++ Swedish BBH18 WTsputum UspA1/UspA2H 42.9/57.3 +++ +++ +++ BBH18 (1 + 2KO) — — + − −BBH18 (1KO) UspA2H 57.3 +++ +++ +++ RH4 WT blood UspA1/UspA2H 37.4/59.9not done ++ ++ RH4 (1 + 2KO) — — − − + *determined using the softwareGapL/ClustalX versus the ATCC25238 UspA2 fragment AA30-540. +++ >50000++ >20000 + >500 − <200

TABLE 9 UspA Expression in the M. catarrhalis strains in Table 8. UspA1UspA2 UspA2H Strains expression expression expression ATCC 25238 Yes YesNo 43617 Yes stop codon No American 2926 Yes No stop codon 2933 Yes YesNo 2912 Yes Yes No 2908 Yes Yes No Finnish 307 Yes Yes No 353 Yes Yes No358 Yes Yes No 216 Yes Yes No Dutch N9 Yes No Yes H2 Yes Yes No F10 YesYes No Norvegian 1 Yes Yes No 13 Yes Yes No 20 Yes Yes No 25 Yes Yes No27 Yes Yes No 36 Yes Yes No Swedish BBH18 WT Yes No Yes BBH18 (1 + 2KO)No No No BBH18 (1KO) No No Yes RH4 WT Yes No Yes RH4 (1 + 2KO) No No No

Example 8: Protection in a Mouse Model of Lung Colonization (MC-001)

Five weeks-old female Balb/c mice (n=8/5 groups) were immunized by theintramuscular route at days 0, 14 and 28 with 50 μl of vaccinecontaining 10 μg of UspA2 construct MC-001 formulated within AS02V. Micewere intranasally challenged at day 42 with 5 10⁵ CFU of variousMoraxella catarrhalis strains. Bacteria were counted in lungs collected0, 3 and 6 hours post-challenge. Differences between groups wereanalysed using the Dunnet test.

As summarised in Table 10, UspA2 construct MC-001 induced a significantprotection against both homologous and heterologous strains, includingthe strain 43617 which does express UspA1 but not UspA2 and the BBH18strain which expresses the chimeric protein UspA2H (constituted of theN-terminal sequence from UspA1 and the C-terminal sequence from UspA2).

TABLE 10 Protective efficacy of UspA2. MC-001 construct. Identity %*versus the vaccinal Log₁₀ cfu/ml UspA sequence Control Vaccine Strainexpressed ATCC25238 group group p value 25238 UspA1 & UspA2 45.2/100 5.23.1 0.01 43617 UspA1 40.1 4.9 3.6 0.01 F10 UspA1 & UspA2 42.9/61.1 4.33.9 0.25 F10 UspA1 & UspA2 42.9/61.1 4.4 3.6 0.01 BBH18 UspA1 & UspA2H42.9/57.3 4.3 3.5 0.01 20 UspA1 & UspA2 33.7/60.6 4.4 3.9 0.02*determined using the GapL/ClustalX software versus the ATCC25238 UspA2fragment AA 30-540 p values in bold are significant (p < 0.05)

Example 9: UspA2 Construct MC-007: Antibody Bactericidal Activity

Bactericidal Assay

Moraxella catarrhalis 25238 was cultivated overnight on Petri dish at37° C.+5% CO₂. Bacteria were transferred in 12 ml HBSS-BSA 0.1% bufferin order to get an OD₆₂₀ of 0.650. Serum samples were heated for 45 minat 56° C. to inactivate the endogenous complement. Serial two-folddilutions of sera in SBA buffer (HBSS-BSA 0.1%) were added on a 96-wellround bottom microtitre plate (25 μl/well). Subsequently, 50 μl of SBAbuffer were added in each well. Then 25 μl of Moraxella catarrhalis25238 strain at 4 10⁴ cfu/mL were added to the wells containing sera andincubated for 15 min at room temperature. Finally 25 μl of freshlythawed baby rabbit complement diluted ⅛ in HBSS-BSA 0.1% were added toreach a final volume of 125 μl. Plates were incubated for 1 h at 37° C.with orbital shaking (210 rpm). The reaction was stopped by laying themicroplate on ice for at least 5 min. After homogenization, variousdilutions of the suspension were added onto chocolate agar plates andincubated for 24 hours at 37° C. with 5% CO₂ and Moraxella colonies werecounted. Eight wells without serum sample were used as bacterialcontrols to determine the number of Moraxella catarrhalis colonies perwell. The mean number of CFU of the control wells was determined andused for the calculation of the killing activity for each serum sample.The bactericidal titers were expressed at the reciprocal dilution ofserum inducing 50% of killing.

Anti-UspA2 antisera generated in mice with UspA2 construct MC-001 orMC-007 were tested in a bactericidal assay using the protocol describedabove against the 25238 Moraxella catarrhalis homologous strain.

As shown in Table 11, the MC-007 UspA2 construct elicited a highbactericidal response, similar to that induced by the MC-001.

TABLE 11 Bactericidal activity of anti-UspA2 MC-001 and MC-007antibodies. Normal mouse sera = sera from mice immunized with AS02Vonly, not with UspA2. Samples Bactericidal titers Normal mouse sera(AS02V) − Mouse Anti-Killed whole cells 25238 ++ Mouse anti-UspA2against +++ UspA2 MC-001 Mouse anti-UspA2 against +++ UspA2 MC-007

Example 10: UspA2 Construct MC-007: Protective Efficacy in a LungChallenge Model

Protection in a Mouse Model of Lung Colonization

Five weeks-old female Balb/c mice (8 mice per group, 5 groups max pertime point) were immunized by the intramuscular route at days 0, 14 and28 with 50 μl of vaccine containing 10 μg of UspA2 construct MC-001formulated with AS02V or MC-007 formulated within AS02V. Mice wereintranasally challenged at day 42 with 5 10⁵ CFU of Moraxellacatarrhalis strain ATCC (a US registered trademark) 25238™. Mice wereimmunized with 10 μg of killed whole cells from Moraxella catarrhalisstrain ATCC (a US registered trademark) 25238™ (as positive control) (M.cat. WC 25238 in FIG. 14) or with AS02V alone (as negative control).Bacteria were counted in lungs collected 0, 3 and 6 hourspost-challenge. Differences between groups were analysed using theDunnet test.

As shown in FIG. 14, both UspA2 constructs were similarly protectiveagainst ATCC (a US registered trademark) strain 25238™.

Example 11: Immunogenicity of UspA2 MC-009 Protein Formulations in Mice

Groups of 25 female Balb/c mice were immunized by the intramuscular (IM)route at days 0, 14 and 28 with 50 μl of the following formulations:

MC-009 (1 μg) AlPO4 (1000 μg/ml)

MC-009 (1 μg) AS04C (AlPO₄/MPL 100/100 per ml)

MC-009 (1 μg) AS01E (QS21/MPL 50/50 per ml)

Anti-IgG levels were determined in individual sera collected at days 28(PII) and 42 (PIII) using the following protocol:

ELISA to Measure Anti-UspA2 Antibodies.

Plates were coated overnight at 4° C. with 100 μl per well of UspA2construct MC-009 at 4 μg/ml in carbonate buffer pH 9.6. The plates werewashed three times with NaCl 0.09% TWEEN (a US registered trademark) 20(polysorbate 20) 0.05%. After washing, serial two fold dilutions of serawere added to microwells in PBS TWEEN (a US registered trademark) 200.05%. The plates were placed at room temperature for 30 minutes withshaking. After washing, anti-mouse IgG antibodies (Jackson 115-035-003)conjugated to peroxydase (100 μl per well) were added, and the plateswere placed at room temperature for 30 minutes with shaking. Plates werewashed as above and the solution of revelation (4 mg of OPDA SigmaP8787) and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH (pH) 4.5) was addedto each well (100 μl/well) for 15 min in darkness. The reaction wasstopped by addition of 50 μl of HCl 1N and the absorbance was read at490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

As shown in FIG. 15, UspA2 induced high antibody levels with eachadjuvant formulation.

Bactericidal Assay

The bactericidal assay was performed against M. catarrhalis strain (ATCC(a US registered trademark) 25238™) expressing a homologous full lengthUspA2 using the following protocol: Moraxella catarrhalis strain ATCC (aUS registered trademark) 25238™ was cultivated overnight on Petri dishat 37° C.+5% CO₂. Bacteria were transferred in 10 ml of BHi (broth heartinfusion) in order to get an OD₆₂₀ of 0.650. Serum samples were heatedfor 45 min at 56° C. to inactivate the endogenous complement. Serialtwo-fold dilutions of sera in SBA buffer (HBSS-BSA 0.1%) were added on a96-well round bottom microtitre plate (25 μl/well). Subsequently, 50 μlof SBA buffer were added in each well. Then 25 μl of Moraxellacatarrhalis strain 25238™ at 4 10³ cfu/mL were added to the wellscontaining sera and incubated for 15 min at room temperature. Finally 25μl of freshly thawed baby rabbit complement diluted ⅛ in HBSS-BSA 0.1%were added to reach a final volume of 125 μl. Plates were incubated for1 h at 37° C. with orbital shaking (210 rpm). The reaction was stoppedby laying the microplate on ice for at least 5 min. A 20 μl aliquot ofeach well of the plate was then transferred into the corresponding wellof a 96-well flat bottom microplate and 50 μl of Mueller HintonBroth-0.9% agar was added to each well. 50 μl of PBS 0.9% agar was addedas a second layer. After 3 hours at 37° C. with 5% CO₂ plates wereincubated overnight at 25° C., Moraxella colonies were counted using anautomated image analysis system (KS 400, Zeiss, Oberkochen, Germany).Eight wells without serum sample were used as bacterial controls todetermine the number of Moraxella per well. The mean number of CFU ofthe control wells was determined and used for the calculation of thekilling activity for each serum sample. The bactericidal titers wereexpressed at the reciprocal dilution of serum inducing 50% of killing.

FIG. 16 illustrates the bactericidal titers induced by UspA2 against ahomologous strain. In this experiment, UspA2 induced high levels ofbactericidal antibodies for each adjuvant formulation. Sera were testedat PIII; five pools of five sera samples were tested.

Example 12: Immunogenicity of UspA2 in Combination with PD and PE-PilANTHi Antigens

Immunization Protocol

Groups of 25 female Balb/c mice were immunized by the intramuscular (IM)route at days 0, 14 and 28 with 50 μl of the following formulations:

UspA2 construct MC-009 (1 μg) AlPO4

UspA2 construct MC-009 (1 μg) AS04C

UspA2 construct MC-009 (1 μg) AS01E

UspA2-PD-PEPilA (UspA2 construct MC-009, PEPilA construct LVL-735) AlPO4(1 ug of each of UspA2, PD and PEPilA; 1000 mg/ml AlPO4)

UspA2-PD-PEPilA (UspA2 construct MC-009, PEPilA construct LVL-735) AS04CAlPO4 (1 ug of each of UspA2, PD and PEPilA; 100/100 per ml AlPO4/MPL)

UspA2-PD-PEPilA (UspA2 construct MC-009, PEPilA construct LVL-735) AS01E(1 ug of each of UspA2, PD and PEPilA; 50/50 per ml QS21/MPL)

ELISA to Measure Anti-UspA2 Antibodies

Anti-UspA2 IgG levels were determined in individual sera collected atdays 28 and 42 using the following protocol.

Plates were coated overnight at 4° C. with 100 μl per well of UspA2construct MC-009 at 4 μg/ml in carbonate buffer pH 9.6. The plates werewashed three times with NaCl 0.09% TWEEN (a US registered trademark) 20(polysorbate 20) 0.05%. After washing, serial two fold dilutions of serawere added to microwells in PBS TWEEN (a US registered trademark) 200.05%. The plates were placed at room temperature for 30 minutes withshaking. After washing, anti-mouse IgG antibodies (Jackson 115-035-003)conjugated to peroxydase (100 μl per well) were added, and the plateswere placed at room temperature for 30 minutes with shaking. Plates werewashed as above and the solution of revelation (4 mg of OPDA SigmaP8787) and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH (pH) 4.5) was addedto each well (100 μl/well) for 15 min in darkness. The reaction wasstopped by addition of 50 μl of HCl 1N and the absorbance was read at490 nm (620 nm for the reference filter). The titers were calculated bythe 4-parameters method using the SOFTMAX (a US registered trademark)Pro software.

ELISA to Measure Anti-PE Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 2 μg/ml ofUspA2 in carbonate buffer pH 9.6. The plates were washed three timeswith NaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

ELISA to Measure Anti-PilA Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 4 μg/ml ofPilA in carbonate buffer pH 9.6. The plates were washed three times withNaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

ELISA to Measure Anti-PD Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 8 μg/ml ofPD in carbonate buffer pH 9.6. The plates were washed three times withNaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

Bactericidal Assay

Bactericidal titres were measured in pooled sera (5 pools/group)collected at day 42 using the following protocol:

Moraxella catarrhalis was cultivated overnight on Petri dish at 37°C.+5% CO2. Bacteria were transferred in 10 ml BHi (broth heart infusion)medium in order to get an OD₆₂₀ of 0.650. Serum samples were heated for45 min at 56° C. to inactivate the endogenous complement. Serial twofold dilutions of sera in SBA buffer (HBSS-BSA 0.1%) were added on a96-well round bottom microtitre plate (25 μl/well).

Subsequently, 50 μl of SBA buffer were added in each well. Then 25 μl ofMoraxella catarrhalis strain 25238 at 4 10³ cfu/mL were added to thewells containing sera and incubated for 15 min at room temperature.Finally 25 μl of freshly thawed baby rabbit complement diluted ⅛ inHBSS-BSA 0.1% were added to reach a final volume of 125 μl. Plates wereincubated for 1 h at 37° C. with orbital shaking (210 rpm). The reactionwas stopped by laying the microplate on ice for at least 5 min. A 20 μlaliquot of each well of the plate was then transferred into thecorresponding well of a 96-well flat bottom microplate and 50 μl ofMueller Hinton Broth-0.9% agar was added to each well. 50 μl of PBS 0.9%agar was added as a second layer. After 3 hours at 37° C. with 5% CO₂plates were incubated overnight at 25° C. Moraxella colonies werecounted using an automated image analysis system (KS 400, Zeiss,Oberkochen, Germany). Eight wells without serum sample were used asbacterial controls to determine the number of Moraxella per well. Themean number of CFU of the control wells was determined and used for thecalculation of the killing activity for each serum sample. Thebactericidal titers were expressed at the reciprocal dilution of seruminducing 50% of killing.

The bactericidal assay was performed against Moraxella catarrhalisstrain 25238™, expressing a homologous UspA2.

A negative impact of the presence of PD and PE-PilA antigens on UspA2IgG levels was observed in AS04C (post III) and AS01E (post II)formulations (FIG. 17). However the impact remained limited (≤2 foldantibody decrease) and was not confirmed in the bactericidal assay (FIG.18). The IgG responses induced against PD, PE and PilA in mice byPE-PEPi1A-UspA2 vaccine are shown in FIG. 19, FIG. 20 and FIG. 21,respectively.

Therefore, UspA2 was immunogenic when combined with PD and PE-PilA.

Example 13: UspA2 Construct MC-009: Immunogenicity of PD and PE-PilANTHi Antigens in Combination with UspA2 in Mice

Immunization Protocol

Groups of 25 female Balb/c mice were immunized by the intramuscular (IM)route at days 0, 14 and 28 with 50 μl of the following formulations:

-   -   PD-PEPilA (1 μg of PD and 1 ug of PEPilA construct LVL-735)        AS01E    -   UspA2-PD-PEPilA (1 μg of UspA2 construct MC-009, PD and PEPilA        construct LVL-735) AS01E

The ELISA IgG levels to PD, PE and PilA were determined in individualsera collected at days 28 (PII) and 42 (PIII).

ELISA to Measure Anti-PE Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 2 μg/ml ofUspA2 in carbonate buffer pH 9.6. The plates were washed three timeswith NaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

ELISA to Measure Anti-PilA Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 4 μg/ml ofPilA in carbonate buffer pH 9.6. The plates were washed three times withNaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

ELISA to Measure Anti-PD Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 8 μg/ml ofPD in carbonate buffer pH 9.6. The plates were washed three times withNaCl 0.09% TWEEN (a US registered trademark) 0.05%. After washing,serial two fold dilution of sera were added to microwells in PBS TWEEN(a US registered trademark) 20 0.05%. The plates were placed at roomtemperature for 30 minutes with shaking. After washing, anti-mouse IgGantibodies (Jackson 115-035-003) conjugated to peroxydase (100 μl perwell) were added, and the plates were placed at room temperature for 30minutes with shaking. Plates were washed as above and the solution ofrevelation (4 mg of OPDA and 5 μl of H₂O₂ in 10 ml of citrate 0.1M PH4.5) was added to each well (100 μl/well) for 15 min in darkness. Thereaction was stopped by addition of 50 μl of HCl 1N and the absorbancewas read at 490 nm (620 nm for the reference filter).

The titers were calculated by the 4-parameters method using the SOFTMAX(a US registered trademark) Pro software.

No major impact of the addition of UspA2 on PD and PEPiIA immunogenicityin AS01E was observed as shown in FIGS. 22, 23 and 24.

Example 14: Safety of a Tetravalent Vaccine Formulation Containing UspA2in a Mouse Moraxella catarrhalis Lung Inflammation Model

To mitigate the risk of inducing undesirable inflammatory responses inthe lungs of COPD patients upon immunization with a candidate vaccineaiming at preventing the exacerbations due to Non-typeable Haemophilusinfluenzae (NTHi) and Moraxella catarrhalis (M. cat.), various animalmodels were developed and used to assess the safety of this vaccine. Theformulation tested contained three NTHi antigens (PD, PE and PilA, withthe two last ones combined as a PEPiIA fusion protein), one M. cat.antigen (UspA2) and the Adjuvant System 01_(E) (AS01_(E)).

Two models were particularly dedicated to evaluate the safety of theUspA2 component of the vaccine.

Model 1:

Objective

This model aimed at assessing the possible induction of undesirableimmune responses in inflamed lungs upon vaccination.

Study Design

C57BI/6 mice were sensitized by three intranasal administrations of 25μg of heat-inactivated M. cat. strain ATCC (a US registered trademark)25238™ whole cells (expressing an UspA2 which is 100% homologous to thevaccine UspA2) at days 0, 7 and 14. This treatment induced in the lungsa perivascular and peribronchiolar inflammation (with formation oflymphoid aggregates), alveolitis, pneumonitis, fibrosis and a strong M.cat. whole cell-specific IL-17⁺ CD4⁺ T cell response, which altogethermimicked the inflammatory process observed in the lungs of COPD patients(except emphysema).

The mice were then vaccinated at day 42 by the intramuscular route with1/10^(th) of human dose of the following formulations:

-   -   PD 10 μg/PEPilA (LVL735 construct, described in WO2012/139225)        10 μg/UspA2 (MC009 construct) 10 μg/AS01_(E)    -   PD 10 μg/PEPilA (LVL735 construct) 10 μg/UspA2 (MC009 construct)        3.3 μg/AS01_(E)    -   AS01_(E) (negative control)    -   PBS (negative control)

To assess the impact of these formulations on the sensitization-inducedlung inflammation:

Mice were daily monitored from day 43 to day 49 to look at mortality andany clinical signs indicating the induction of adverse events(prostration, piloerection, hunched position).

A histological analysis of the lungs was performed at days 2, 7 and 14post-vaccination (with 5 mice per group and time-point) to look at apossible aggravation of the inflammation.

The induction of potentially undesirable T cell responses was evaluatedon pools of lungs collected at days 7 and 14 post-vaccination (with 4pools/group/time-point and the lungs of 3 mice per pool). The lung Tcells were re-stimulated overnight either with UspA2 peptides,heat-inactivated M. cat. whole cells (WC) or medium (as a negativecontrol) and then analyzed by flow cytometry for the expression of CD5,CD4, CD8, IL-17, IL-13, TNFα and IFNγ.

-   -   Results    -   No mortality or adverse event was reported.    -   Lung histology (FIGS. 25 to 29):        -   The alterations observed in the lungs were similar in            severity in all groups and characterized by slight to            moderate perivascular/bronchiolar mononuclear cell            infiltrates.        -   No alveolitis and/or pneumonitis related to vaccination were            observed.    -   T cell response:        -   Strong CD4⁺ T cell responses (mainly IL-17 and TNFα            producing cells) were measured in the lungs upon            re-stimulation with WC, but regardless of the formulation            administered (vaccines or adjuvant alone or PBS) (FIGS. 30            to 33). Low or no lung CD8⁺ T cell responses were observed            (data not shown).        -   No detectable T cell response was re-stimulated by UspA2            peptides, whatever the group, indicating that no            UspA2-specific response was primed or boosted            post-vaccination (data not shown).

Model 2:

Objective

This model aimed at assessing the possible induction of undesirableimmune responses in inflamed lungs upon vaccination and M. cat.challenge.

Study Design

C57BI/6 mice were successively:

-   -   Sensitized by three intranasal administrations of 25 μg of        heat-inactivated M. cat. strain 25238 WC (expressing an UspA2        which is 100% homologous to the vaccine UspA2) at days 0, 7 and        14 (as in Model 1).    -   Vaccinated at day 42 by the intramuscular route with 1/10^(th)        of human dose of the following formulations (as in Model 1):        -   PD (10 μg/PEPilA (LVL735 construct) 10 μg/UspA2 (MC009            construct) 10 μg/AS01_(E)        -   PD 10 μg/PEPilA (LVL735 construct) 10 μg/UspA2 (MC009            construct) 3.3 μg/AS01_(E)        -   AS01_(E) (negative control)        -   PBS (negative control)    -   Challenged by one intranasal administration of 25 μg of        heat-inactivated M. cat. strain F10 WC (expressing an UspA2        which shares 53% homology with the vaccine UspA2) or by one        intranasal administration of PBS as a control, both at day 56.        The challenge strain was different from the sensitization strain        to mimic the situation observed in COPD patients who experience        new exacerbations due to newly acquired M. cat. strains.

To assess the impact of vaccination and challenge on thesensitization-induced lung inflammation:

-   -   Mice were daily monitored from day 43 to day 63 to look at        mortality and any clinical signs indicating the induction of        adverse events (prostration, piloerection, hunched position).    -   The induction of potentially undesirable T cell responses was        evaluated on pools of lungs collected at days 7 and 14        post-challenge (with 4 pools/group/time-point and the lungs of 3        mice per pool). The lung T cells were re-stimulated overnight        either with UspA2 peptides, heat-inactivated M. cat. F10 WC or        medium (as a negative control) and then analyzed by flow        cytometry for the expression of CD5, CD4, CD8, IL-17, IL-13,        TNFα and IFNγ.

Results

-   -   No mortality or adverse event was reported.    -   T cell response:        -   Strong post-challenge CD4⁺ T cell responses (mainly IL-17            and TNFα producing cells) were measured in the lungs upon            re-stimulation with F10 WC, regardless of the formulation            administered (vaccines or adjuvant alone or PBS) (FIGS. 34            to 37). Not surprisingly, these responses were higher in            mice challenged with inactivated bacteria than in mice            challenged with PBS. Whatever the challenge, low or no lung            CD8⁺ T cell responses were observed (data not shown).        -   No detectable T cell response was re-stimulated by UspA2            peptides, whatever the group, indicating that no            UspA2-specific response was primed or boosted post-challenge            (data not shown).

CONCLUSION

The PD/PEPilA/UspA2/AS01_(E) formulations tested and more specificallythe UspA2 component of these vaccines were shown safe in a mouse M. cat.lung inflammation model.

The invention claimed is:
 1. A protein of formula I:A-(R₁)_(m)—(B)_(n)  (formula I) wherein: A is an immunogenic fragment ofUspA2 selected from the group consisting of amino acids 30-540 of SEQ IDNO: 1 (SEQ ID NO: 39), amino acids 31-540 of SEQ ID NO: 1 (SEQ ID NO:40), amino acids 30-519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids30-564 of SEQ ID NO: 1 (SEQ ID NO: 42) and amino acids 31-564 of SEQ IDNO: 1 (SEQ ID NO: 43); R₁ is AS (alanine serine); m is 0, 1 or 2; B ishistidine; and n is 0, 1, 2, 3, 4, 5, or 6, wherein when m is 0, n isnot
 0. 2. The protein according to claim 1 further comprising amethionine at the amino terminal.
 3. An immunogenic compositioncomprising the protein according to claim 2, wherein A is SEQ ID NO: 43,and a pharmaceutically acceptable excipient.
 4. The immunogeniccomposition according to claim 3, further comprising an antigen fromHaemophilus influenzae.
 5. The immunogenic composition according toclaim 4, wherein the antigen from Haemophilus influenzae is Protein D.6. The immunogenic composition according to claim 5, further comprisingProtein E from Haemophilus influenzae.
 7. The immunogenic compositionaccording to claim 6, further comprising Pilin A from Haemophilusinfluenzae.
 8. The immunogenic composition according to claim 7, whereinthe Protein E and Pilin A from Haemophilus influenzae is present as aPE-PilA fusion protein.
 9. The immunogenic composition according toclaim 3 further comprising an adjuvant.
 10. The immunogenic compositionaccording to claim 8, wherein the PE-PIIA fusion protein is LVL735. 11.The immunogenic composition according to claim 9, wherein the adjuvantis AS01E.
 12. A protein of formula I:A-(R₁)_(m)—(B)_(n)  (formula I) wherein: A is an immunogenic fragment ofUspA2 having amino acids 31-564 of SEQ ID NO: 1 (SEQ ID NO: 43); R₁ isan amino acid; m is 0, 1 or 2, B is histidine; and n is 0, 1, 2, 3, 4,5, or 6, wherein when m is 0, n is not
 0. 13. The protein according toclaim 12, further comprising a methionine at the amino terminal.
 14. Animmunogenic composition comprising the protein according to claim 13 anda pharmaceutically acceptable excipient.
 15. The immunogenic compositionaccording to claim 14, further comprising an antigen from Haemophilusinfluenzae.
 16. The immunogenic composition according to claim 15,wherein the antigen from Haemophilus influenzae is Protein D.
 17. Theimmunogenic composition according to claim 16, further comprisingProtein E from Haemophilus influenzae.
 18. The immunogenic compositionaccording to claim 17, further comprising Pilin A from Haemophilusinfluenzae.
 19. The Immunogenic composition according to claim 18,wherein the Protein E and Pilin A from Haemophilus influenzae is aPE-PilA fusion protein.
 20. The immunogenic composition according toclaim 15, further comprising an adjuvant.
 21. The immunogeniccomposition according to claim 19, wherein the PE-PIIA fusion protein isLVL735.
 22. The immunogenic composition according to claim 20, whereinthe adjuvant is AS01E.